Methods and compositions for modulating immunity

ABSTRACT

Methods and compositions for inducing immune suppression are disclosed. The methods involve administering an effective amount of a CD200 protein or a nucleic acid encoding a CD200 protein. The methods are useful in preventing graft rejection, fetal loss, autoimmune disease, and allergies. Methods and compositions for preventing immune suppression are also disclosed. The methods involve administering an effective amount of an agent that inhibits CD200. Such methods are useful in treating cancer.

RELATED APPLICATIONS

This application is a continuation of U.S. application no. 11/736,632filed Apr. 18, 2007 (now allowed), which is a continuation of U.S.patent application Ser. No. 10/850,562 filed May 21, 2004 (now allowed),which is a continuation-in-part of (1) U.S. patent application Ser. No.09/948,725 filed Sep. 10, 2001 (now Pat. No. 6,984,625); (2) U.S. patentapplication Ser. No. 09/917,278 filed Jul. 30, 2001 (now U.S. Pat. No.6,955,811); and (3) U.S. patent application Ser. No. 09/934,634 filedAug. 23, 2001 (now U.S. Pat. No. 6,749,854), each of which are acontinuation-in-part of U.S. application Ser. No. 09/570,367 filed May5, 1998 (now U.S. Pat. No. 6,338,851) which is a continuation ofPCT/CA98/01038 filed Nov. 6, 1998 (which designated the U.S.) whichclaims the benefit of U.S. provisional application Ser. No. 60/064,764filed Nov. 7, 1997 (now abandoned). U.S. patent application Ser. No.09/917,278 (now U.S. Pat. No. 6,955,811) also claimed the benefit ofU.S. provisional Ser. No. 60/222,725 filed Aug. 3, 2000 (now abandoned).All of the prior applications are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for modulatingan immune response. The invention includes the use of the protein CD200to suppress an immune response and the use of an agent that inhibitsCD200 to prevent immune suppression.

BACKGROUND OF THE INVENTION

The immune system protects the body from infectious agents and diseaseand is critical to our survival. However, in certain instances, theimmune system can be the cause of illness. One example is in autoimmunedisease wherein the immune system attacks its own host tissues, in manyinstances causing debilitating illness and sometimes resulting in death.Examples of autoimmune diseases include multiple sclerosis, type 1insulin-dependent diabetes mellitus, lupus erythematosus and arthritis.A second example where the immune system can cause illness is duringtissue or organ transplantation. Except in the cases of geneticallyidentical animals, such as monozygotic twins, tissue and organtransplants are rejected by the recipient's immune system as foreign.The immune reaction against transplants is even more pronounced intransplantation across species or xenotransplantation. A third examplewhere the immune system harms the host is during an allergic reactionwhere the immune system is activated by a generally innocuous antigencausing inflammation and in some cases tissue damage.

In order to inhibit the detrimental immune reactions duringtransplantation, autoimmune disease and allergic reactions,immunosuppressive drugs (such as cyclosporin A, tacrolimus, andcorticosteroids) or antibody therapies (such as anti-T cell antibodies)are generally administered. Unfortunately, these non-specific modes ofimmunosuppression generally have undesirable side effects. For example,cyclosporin may cause decreased renal function, hypertension, toxicityand it must be administered for the life of the patient. Corticosteroidsmay cause decreased resistance to infection, painful arthritis,osteoporosis and cataracts. The anti-T cell antibodies may cause fever,hypertension, diarrhea or sterile meningitis and are quite expensive.

In view of the problems associated with immunosuppression, there hasbeen an interest in developing methods or therapies that induceunresponsiveness or tolerance in the host to a transplant, to “self”tissues in autoimmune disease and to harmless antigens associated withallergies. The inventor has been studying the mechanisms involved intransplant rejection and has developed methods for inducing a state ofantigen-specific immunological tolerance in transplantation. Inparticular, in animal allograft models, the inventor has demonstratedthat graft survival can been increased if the recipient animal is givena pre-transplant infusion via the portal vein of irradiated spleen cellsfrom the donor animal. In contrast, a pre-transplant infusion via thetail vein does not prolong graft survival.

Understanding the molecular mechanisms involved in the induction oftolerance following portal-venous (pv) immunization may lead to thedevelopment of methods of inducing immune tolerance that may be usefulin transplant, autoimmune disease and allergies.

SUMMARY OF THE INVENTION

The present inventors have shown that CD200, a molecule with previouslyunknown function belonging to the Ig superfamily, is an immunesuppressant. The inventors have shown that administering antibodies toCD200 inhibited the graft survival generally seen followingpre-transplant pv immunization. The inventors have also shown that thereis a negative association between levels of CD200 and the risk of fetalloss. In particular, the inventors have shown administering CD200reduced fetal loss rates while inhibiting CD200 reversed the effect. Theinventors have also shown that administering CD200 inhibits thedevelopment of autoimmune diseases such as arthritis. The inventors havefurther shown that CD200 inhibits cytotoxic cells and IL-2 productionand induces IL-4 production. The inventors have also shown that CD200 isresponsible for promoting tumor metastases and inhibiting CD200 reducestumor cell growth. All of these results demonstrate that CD200 isinvolved in immune suppression.

Consequently, in one aspect, the present invention provides a method ofsuppressing an immune response comprising administering an effectiveamount of a CD200 protein, or a fragment thereof, or a nucleic acidsequence encoding a CD200 protein, or a fragment thereof, to an animalin need of such treatment.

In one embodiment, the present invention provides a method of preventingor inhibiting an autoimmune disease comprising administering aneffective amount of a CD200 protein or a nucleic acid sequence encodinga CD200 protein to an animal in need thereof.

In another embodiment, the present invention provides a method ofpreventing or inhibiting fetal loss comprising administering aneffective amount of a CD200 protein or a nucleic acid sequence encodinga CD200 protein to an animal in need thereof.

In a further embodiment, the present invention provides a method ofinducing tumor cell growth or metastases comprising administering aneffective amount of a CD200 protein or a nucleic acid sequence encodinga CD200 protein to an animal in need thereof.

In another embodiment, the present invention provides a method ofsuppressing an immune response to a transplanted organ, tissue or cellcomprising administering an effective amount of a CD200 protein or anucleic acid sequence encoding a CD200 protein to an animal in needthereof.

In yet another embodiment, the present invention provides a method ofpreventing or inhibiting allergy comprising administering an effectiveamount of a CD200 protein or a nucleic acid sequence encoding a CD200protein to an animal in need thereof.

The invention also includes pharmaceutical compositions containing CD200proteins or nucleic acids encoding CD200 proteins for use in suppressingan immune response.

The inventors have shown that inhibiting CD200 is useful in preventingimmune suppression and is useful in treating cancer and inducing fetalloss.

Therefore, in another aspect, the present invention provides a method ofpreventing immune suppression or inducing an immune response comprisingadministering an effective amount of an agent that inhibits CD200 to ananimal in need thereof. In a preferred embodiment the CD200 inhibitor isan antibody that binds CD200 or an antisense oligonucleotide thatinhibits the expression of CD200.

In one embodiment, the present invention provides a method of inhibitingthe growth of a tumor cell comprising administering an effective amountof an agent that inhibits CD200 to a cell or animal in need thereof.

In another embodiment, the present invention provides a method ofinducing fetal loss comprising administering an effective amount of anagent that inhibits CD200 to an animal in need thereof.

The invention also includes pharmaceutical compositions containing aCD200 inhibitor for use in inducing or augmenting an immune response.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1 illustrates PCR validation of suppressive subtractivehybridization using β-actin primers.

FIG. 2 illustrates PCR validation of suppressive subtractivehybridization using IL-10 primers.

FIG. 3 is an autoradiograph using ³²P-labeled probes from 4 clonesobtained from the subtractive hybridization process.

FIG. 4 is flow cytometry profile of spleen adherent cells.

FIGS. 5A and B are Western Blots illustrating the increased expressionof OX-2 antigen after pv immunization. FIG. 5A shows staining with acontrol mouse antibody, anti-mouse CD8a. FIG. 5B shows staining withanti-rat MRC OX-2.

FIG. 6 is a graft showing percent survival versus days post renaltransplantation.

FIG. 7 shows the cDNA sequence of rat (SEQ ID NO:20), mouse (SEQ IDNO:22) and human MRC OX-2 (SEQ ID NO:18).

FIG. 8 shows the deduced protein sequence of rat (SEQ ID NO:21), mouse(SEQ ID NO:2) and human MRC OX-2 (SEQ ID NO:19) protein.

FIGS. 9A and 9B are bar graphs showing cytokine production and cellproliferation following stimulation by allogeneic DC using hepatic NPMC.

FIGS. 10A, 10B and 10C are bar graphs showing inhibition of cellproliferation and cytokine production by hepatic NPMC.

FIG. 11 is a bar graph analysis of FACS data showing OX-2 expression ina subpopulation of NPC.

FIG. 12 shows PCR analysis mRNA expression of B7-1, B7-2 and OX-2 invarious hepatic NPMC cell fractions.

FIGS. 13A and 13B are bar graphs showing proliferation and cytokineproduction by NPMC from Flt3L treated mice.

FIG. 14 is a bar graph showing cytokines produced from C3H mice withC57BL16 renal allografts and NPC from Flt3 treated C57BL16 donors.

FIG. 15 is a graph showing inhibition of graft rejection with NPC fromFlt3 treated mice.

FIG. 16 is a graph showing that anti-OX-2 reverses inhibition by NPC.The effect of anti-B7-1, anti-B7-2 and anti-OX-2 on primaryallostimulation is shown.

FIG. 17 is a graph showing that anti-OX-2 mAb reverses inhibition by NPCand inhibits the development of immunoregulatory cells.

FIG. 18A is a photograph showing in situ hybridization with antisenseOX-2 in a day 8.5 implantation site from a mouse CBA/JxDBA/2 that issusceptible to fetal loss.

FIG. 18B is a photograph showing in situ hybridization with antisenseOX-2 in a day 8.5 implantation site from a mouse DBA/2XCBA/J that is notsusceptible to spontaneous fetal loss.

FIG. 19 is a graph showing CD200Fc inhibits induction ofcollagen-induced arthritis in mice. Groups of 7 DBA/1 mice wereimmunized with 100 μg bovine collagen type-II in CFA, followed byreimmunization at day 18 with 100 μg collagen in ICFA (see Materials andMethods in Example 8). Thereafter mice received 15 μg iv of normal mouseIg or CD200Fc, at 3 day intervals until sacrifice. Scoring of disease inindividual mice is described elsewhere (Gorczynski, Clin. Immunol; 2001,in press). Data are arithmetic means (±SEM) of the 7 scores/group ateach time point.

FIG. 20 is a bar graph showing ³HTdR incorporation in PLN cellsharvested (at 36 days) from the groups of 7 mice treated in vivo withCD200Fc (or saline) following collagen immunization, as in FIG. 19, andrestimulated in vitro with collagen or BALB/c spleen stimulator cells.No additional CD200Fc was included in vitro. For both sets of controlmice (PBS injected and normal mouse Ig-treated groups), cells werefurther subdivided into those taken from mice with a mean arthriticscore ≧6 (5/7 respectively) and ≦5 (2/7). All CD200Fc treated mice hadarthritic scores <5. Background proliferation in cells from all groupswithout stimulation was in the same range (1250±450 cpm).

FIG. 21 is a bar graph showing CD200Fc given in vivo to mice immunizedwith bovine collagen (see FIGS. 19 and 20) blocks development ofincreased serum TNFα and IFNγ. Once again data for the two groups ofcontrol animals (untreated or control Ig treated) are subdivided intoresults from animals with arthritic score ≧6 or ≦5 respectively. Serumwas assessed at 36 days post collagen immunization. Background serumTNFα and IFNγ cytokine levels (pg/ml) in a group of 5 normal DBA/1 mice(non-immunized) was 25±7.7 and 32±10 respectively.

FIG. 22 is a bar graph showing inhibition of sensitization for furtherTNFα and IFNγ production in vitro using PLN cells of mice treated invivo with CD200Fc and immunized with collagen as in FIGS. 19-21. Cellswere taken from all groups at 36 days post collagen immunization, andwere stimulated in vitro with either collagen or BALB/c cells. Nofurther CD200Fc was included in the cultures. Cytokines were assayed inthe culture supernatants by ELISA at 40 hrs. Control cytokineproduction, averaged over all groups in the absence of stimulation, forTNFα/IFNγ was 65±15 and 110±20 pg/ml respectively.

FIG. 23 is a bar graph showing CD200Fc alters quantitative andqualitative production of autoantibody to collagen type-II in collagenimmunized mice. Data show total serum IgG and different IgG isotypelevels at 36 days of sacrifice in the 3 groups of mice documented inFIGS. 19-22. All data points represent arithmetic means (±SD) of 7mice/group. Similar data were obtained in a repeat study.

FIG. 24 is a graph showing inhibition of EL4 or C1498 tumor growth inC3H bone marrow reconstituted C57BL/6 mice. Groups of 6 BL/6 micereceived 20×10⁶ T-depleted BL/6 or C3H bone marrow cells 24 hrsfollowing cyclophosphamide treatment. 5×10⁶ EL4 or 5×10⁵ C1498 tumorcells were injected 28 days later into these mice, and control BL/6 orC3H mice. >85% of PBL from C3H reconstituted BL/6 were stained by FITCanti-H2K^(k) mAb at this time.

FIG. 25 is a graph showing EL4 tumor growth in BL/6 mice immunizedtwice, at 14 day intervals, with 5×10⁶ EL4 cells transfected to expressCD80 or CD86. 5×10⁶ EL4 cells were injected as tumor challenge 10 daysafter the last immunization.

FIG. 26 is a graph showing suppression of growth inhibition in C57BL/6BMT recipients of EL4 or C1498 tumor cells (see FIG. 19) following 4weekly infusions of 100 μg/mouse anti-CD4 or anti-CD8 mAb, beginning onthe day of BMT (tumor cells were injected at 28 days post BMT). Data areshown for 6 mice/group.

FIG. 27 is a graph showing inhibition of immunity to EL4 or C1498 tumorchallenge following infusion of CD200Fc in C57BL/6 mice reconstitutedwith C3H bone marrow—see FIG. 24 and text for more details.Cyclophosphamide treated BL/6 mice received bone marrow rescue withT-depleted C3H or BL/6 cells. 28 days later all mice, and groups ofcontrol normal C3H mice, received ip injection with 5×10⁶ EL4 or 5×10⁵C1498 tumor cells. Bone marrow reconstituted mice received further ivinfusion of normal mouse IgG or CD200Fc (10 μg/mouse/injection) 5 timesat 2 day intervals beginning on the day of tumor injection.

FIG. 28 is a graph showing inhibition of immunity to EL4 tumor cells inEL4-CD80 immunized BL/6 mice using CD200Fc—see FIG. 25 and text fordetails. Mice received iv infusion of control IgG or CD200Fc asdescribed in FIG. 27.

FIG. 29 is a graph showing improved tumor immunity in EL4-CD86 orC1498-CD86 immunized C57BL/6 mice following infusion of anti-CD200 mAb.See legend to FIG. 25 and text for more details. Where shown, groups ofmice received iv infusion of anti-CD200, 100 mg/mouse, on 3 occasions at3 day intervals beginning on the day of tumor injection.

FIG. 30 is a graph showing log 10 relative concentrations of CD200 mRNAscompared with standardized control mRNA. All samples were firstnormalized for equivalent concentrations of GAPDH mRNA. Values shownrepresent arithmetic means±SD for 3 individual samples for each timepoint. Mice were preimmunized with CD80/CD86-transfected tumor cells asdescribed in the text.

FIG. 31 is a graph showing increased inhibition of tumor immunity usinginfusion of CD200Fc with CD200^(r+) cells in C57BL/6 recipients of C3Hbone marrow. In this experiment some mice received not only CD200Fc withEL4 or C1498 tumor, but in addition a lymphocyte-depleted,LPS-stimulated, macrophage population stained (>65%) with anti-CD200^(r)mAb (2F9).

FIG. 32 is a graph showing combinations of CD200Fc and anti-CD4 oranti-CD8 mAb produce increased suppression of tumor growth inhibition inC57BL/6 recipients of C3H BMT. Groups of 6 mice received weekly ivinfusions of 100 μg anti-T cell mab or 5 iv infusions of 10 μg/mouseCD200Fc, alone or in combination, beginning on the day of tumorinjection (28 days post BMT).

FIG. 33 is a graph showing effect of combined CD200Fc and anti-CD4 oranti-CD8 mAb on suppression of EL4 tumor growth inhibition in C57BL/6recipients preimmunized with EL4-CD80 transfected cells (see FIG. 20).Data are shown for groups of 6 mice/group. Weekly iv infusions of 100 μganti-T cell mab or 5 iv infusions of 10 μg/mouse CD200Fc, alone or incombination, were begun on the day of tumor injection (10 days after thefinal immunization with EL4-CD80 cells).

FIGS. 34A and B are bar graphs showing the median number of lung nodulesin mice receiving allogeneic blood by tail vein.

FIGS. 35A and B are bar graphs showing the number of lung nodules in thepresence of anti-OX2 in mice receiving allogeneic blood by tail vein.

FIGS. 36A and B are bar graphs showing the number of lung nodules in thepresence of anti-OX-2, DEC205 or anti-CD11c in mice receiving allogeneicblood by tail vein.

FIG. 37 is a graph showing the effect of anti-OX-2 monoclonal antibody3B6 on spontaneous resorption (abortion) rates. Error bars show 1standard deviation. A minimum of 20 implants was scored for eachgroup. * significant increase in abortion rate, P<0.02.

FIG. 38 is a graph showing effect of OX2 immunoadhesion (OX2:Fc) onspontaneous resorption (abortion) rates or renal allograft rejection.Error bars show 1 standard deviation. A minimum of 45 implants wasscored for each group of pregnant mice (mice received a single dose ofOX2:Fc at day 6.5; 10 mice/group were used for renal allografts, andmice received multiple iv injections of OX2:Fc, beginning on the day oftransplant, and at 2 day intervals thereafter. * significant decrease inabortion rate, or increased graft survival, relative to controls.

FIG. 39 shows the importance of CD200 (OX-2) in rescuing potentiallydoomed embryos which are low in OX-2 mRNA expression by the in situhybridisation result.

FIG. 40 shows the molecule size ladder (lane 1), trophoblast with fulllength primers (lane 2) and exon 3 primers (lane 3). The full length andshorter mRNA OX-2 transcripts are seen. Lane 4 (corresponding to lane 2)and lane 5 (corresponding to lane 3) represent negative result obtainedwith CD9⁺ stromal cells.

FIG. 41 shows result using antibody detecting CK5, 6, 8, 17; similarresults have been obtained using FITC-anti-CK18. The upper left panelshows the forward and side scatter pattern. FITC and PE isotype controlsused to set gates is shown below on left. Upper right panel showscytokeratin-positive cells, the majority of which were also OX-2⁺. Asignificant population of CK-OX-2⁺⁺ cells was also noted. The identityof this population has not been established. On the right, the middlepanel shows staining profile for OX-2 for the CK-and CK⁺ populations,and the lower panel shows the scatter. Clearly these populations havedifferent properties. Velocity sedimentation separation indicates bothpopulations may have OX-2-dependent immunoregulatory activity.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have identified genes that show an increase inexpression following portal venous immunization. These genes play a rolein the development of immune suppression or tolerance and may be usefulin developing therapies for the prevention and treatment of transplantrejection, fetal loss, autoimmune disease, allergies and cancer.

Using suppression subtractive hybridization (SSH), the inventor hasisolated a clone that is preferentially expressed in mice receivingallogenic renal grafts along with pre-transplant donor-specificimmunization and that encodes the protein CD200 (previously known asOX-2). The CD200 protein (also known as MRC CD200 in rat) was describedas a 41 Kd-47 Kd glycoprotein which is expressed on the cell surface ofthymocytes, follicular dendritic cells and endothelium, B cells andneuronal cells. Differences in apparent size of the molecule indifferent tissues is probably a function of differential glycosylation.The function of the molecule was previously unknown, but DNA and aminoacid sequence analysis shows it has a high degree of homology tomolecules of the immunoglobulin gene family, which includes moleculesimportant in lymphocyte antigen recognition and cell-cell interaction(e.g. CD4, CD8, ICAMs, VCAMs), as well as adhesion receptor molecules(NCAMs) in the nervous system. Members of the immunoglobulin superfamilyare distinct from other molecules of the integrin and selectin families,which, at least within the immune system, also seem to play criticalrole in cell recognition, migration and even development of thelymphocyte recognition repertoire (by regulating intra-thymic selectionevents). It has become increasingly evident that molecules of thesedifferent families play an important role in human disease.

The inventor has shown that administering antibodies to CD200 inhibitedthe graft survival generally seen following pre-transplant pvimmunization. The inventors have also shown that administering CD200inhibits autoimmune disease. The inventors have further shown that thereis negative association between levels of CD200 and risk of fetal lossand that administering CD200 prevents fetal loss and inhibiting CD200causes fetal loss. The inventors have further shown that CD200 inhibitscytotoxic cells and IL-2 production and induces IL-4 production. Theinventors have further shown that CD200 promotes tumor cell growth andinhibiting CD200 inhibits tumor cell growth. The data supports the roleof CD200 in immune suppression.

A. Therapeutic Methods

1. Inducing Immune Suppression

In one aspect, the present invention provides a method of suppressing animmune response comprising administering an effective amount of a CD200protein or a nucleic acid sequence encoding a CD200 protein to an animalin need of such treatment. The invention includes a use of an effectiveamount of a CD200 protein or a nucleic acid sequence encoding a CD200protein to suppress an immune response.

The term “CD200 protein” includes CD200 from any species or source andincludes a full length CD200 protein as well as fragments or portions ofthe protein. The term “CD200” was previously referred to as “OX-2”although there has been a change in nomenclature. Both “CD200” and“OX-2” may be used interchangeably in the application. Preferredfragments or portions of the CD200 or OX-2 protein are those that aresufficient to suppress an immune response. Determining whether aparticular CD200 protein or fragments thereof can suppress an immuneresponse can be assessing using known in vitro immune assays including,but not limited to, inhibiting a mixed leucocyte reaction; inhibiting acytotoxic T cell response; inhibiting interleukin-2 production;inhibiting IFNγ production; inhibiting a Th1 cytokine profile; inducingIL-4 production; inducing TGFβ production; inducing IL-10 production;inducing a Th2 cytokine profile; inhibiting immunoglobulin production;altering serum immunoglobulin isotype profiles (from those associatedwith Th1 type immunity-in the mouse, IgG1 and IgG2a, to those associatedwith Th2 type immunity-in the mouse, IgG2b, IgG3); and any other assaythat would be known to one of skill in the art to be useful in detectingimmune suppression.

The term “administering a CD200 protein” includes both theadministration of the CD200 protein as well as the administration of anucleic acid sequence encoding a CD200 protein. In the latter case, theCD200 protein is produced in vivo in the animal.

In a preferred embodiment, the CD200 protein is prepared andadministered as a soluble fusion protein. The fusion protein may containthe extracellular domain of CD200 linked to an immunoglobulin (Ig) FcRegion. The CD200 fusion may be prepared using techniques known in theart. Generally, a DNA sequence encoding the extracellular domain ofCD200 is linked to a DNA sequence encoding the Fc of the Ig andexpressed in an appropriate expression system where the CD200-FcIgfusion protein is produced.

The CD200 protein may be obtained from known sources or prepared usingrecombinant DNA techniques. The protein may have any of the knownpublished sequences for CD200 or OX-2. The sequences can be obtainedfrom GenBank. The human sequence has accession no. M17226×0523; the ratsequence has accession no. X01785; and the mouse sequence has accessionno. AF029214. The nucleic acid and protein sequences of CD200 (OX-2)from human, mouse and rat are also shown in FIGS. 7 and 8 and in SEQ IDNos:18, 22 and 20 (nucleic acid) and SEQ ID Nos:19, 21 and 2 (protein).

The CD200 protein may also be modified to contain amino acidsubstitutions, insertions and/or deletions that do not alter theimmunosuppressive properties of the protein. Conserved amino acidsubstitutions involve replacing one or more amino acids of the CD200amino acid sequence with amino acids of similar charge, size, and/orhydrophobicity characteristics. When only conserved substitutions aremade the resulting analog should be functionally equivalent to the CD200protein. Non-conserved substitutions involve replacing one or more aminoacids of the CD200 amino acid sequence with one or more amino acidswhich possess dissimilar charge, size, and/or hydrophobicitycharacteristics.

The CD200 protein may be modified to make it more therapeuticallyeffective or suitable. For example, the CD200 protein may be cyclized ascyclization allows a peptide to assume a more favourable conformation.Cyclization of the CD200 peptides may be achieved using techniques knownin the art. In particular, disulphide bonds may be formed between twoappropriately spaced components having free sulfhydryl groups. The bondsmay be formed between side chains of amino acids, non-amino acidcomponents or a combination of the two. In addition, the CD200 proteinor peptides of the present invention may be converted intopharmaceutical salts by reacting with inorganic acids includinghydrochloric acid, sulphuric acid, hydrobromic acid, phosphoric acid,etc., or organic acids including formic acid, acetic acid, propionicacid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinicacid, malic acid, tartaric acid, citric acid, benzoic acid, salicylicacid, benzenesulphonic acid, and toluenesulphonic acids.

Administration of an “effective amount” of the CD200 protein and nucleicacid of the present invention is defined as an amount effective, atdosages and for periods of time necessary to achieve the desired result.The effective amount of the CD200 protein or nucleic acid of theinvention may vary according to factors such as the disease state, age,sex, and weight of the animal. Dosage regima may be adjusted to providethe optimum therapeutic response. For example, several divided doses maybe administered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

The term “animal” as used herein includes all members of the animalkingdom including humans.

(i) Autoimmune Disease

As stated previously, the method of the present invention may be used totreat or prevent autoimmune disease. In an autoimmune disease, theimmune system of the host fails to recognize a particular antigen as“self” and an immune reaction is mounted against the host's tissuesexpressing the antigen. Normally, the immune system is tolerant to itsown host's tissues and autoimmunity can be thought of as a breakdown inthe immune tolerance system.

Accordingly, in a further embodiment, the present invention provides amethod of preventing or treating an autoimmune disease comprisingadministering an effective amount of a CD200 protein or fragmentthereof, or a nucleic acid sequence encoding a CD200 protein or fragmentthereof to an animal having, suspected of having, or susceptible tohaving an autoimmune disease. The invention includes a use of aneffective amount of a CD200 protein on a nucleic acid molecule encodinga CD200 protein to prevent or inhibit an autoimmune disease.

The term “treatment or treating” as used herein means an approach forobtaining beneficial or desired results, including clinical results.Beneficial or desired clinical results can include, but are not limitedto, alleviation or amelioration of one or more symptoms or conditions,diminishment of extent of disease, stabilized (i.e. not worsening) stateof disease, preventing spread of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treating” can also mean prolonging survival as comparedto expected survival if not receiving treatment.

Autoimmune diseases that may be treated or prevented according to thepresent invention include, but are not limited to, arthritis, type 1insulin-dependent diabetes mellitus, adult respiratory distresssyndrome, inflammatory bowel disease, dermatitis, meningitis, thromboticthrombocytopenic purpura, Sjögren's syndrome, encephalitis, uveitis,leukocyte adhesion deficiency, rheumatoid arthritis, rheumatic fever,Reiter's syndrome, psoriatic arthritis, progressive systemic sclerosis,primary biliary cirrhosis, pemphigus, pemphigoid, necrotizingvasculitis, myasthenia gravis, multiple sclerosis, lupus erythematosus,polymyositis, sarcoidosis, granulomatosis, vasculitis, perniciousanemia, CNS inflammatory disorder, antigen-antibody complex mediateddiseases, autoimmune haemolytic anemia, Hashimoto's thyroiditis, Gravesdisease, habitual spontaneous abortions, Reynard's syndrome,glomerulonephritis, dermatomyositis, chronic active hepatitis, celiacdisease, tissue specific autoimmunity, degenerative autoimmunity delayedhypersensitivities, autoimmune complications of AIDS, atrophicgastritis, ankylosing spondylitis and Addison's disease.

One of skill in the art can determine whether or not a particular CD200or fragment thereof is useful in preventing autoimmune disease. Asmentioned previously, one of skill in the art can readily test a CD200or CD200 fragment for its ability to suppress an immune response usingknown in vitro assays. In addition the CD200 or CD200 fragment can alsobe tested for its ability to prevent autoimmune in an animal model. Forexample, one could use the model described in Example 8 wherein theability of CD200 to prevent collagen induced arthritis is assessed.Further, many other autoimmune animal models are available, including,but not limited to, experimental allergic encephalomyelitis which is ananimal model for multiple sclerosis, animal models of inflammatory boweldisease (induced by immunization, or developing in cytokine-knockoutmice), and models of autoimmune myocarditis and inflammatory eyedisease.

(ii) Fertility

The present inventors have shown that there is an association betweenlevels of CD200 expression and fertility. In particular the inventor hasshown that low levels (or no levels) of CD200 is related to fetal loss.Further, administering a CD200:Fc fusion protein prevented fetal loss.

Accordingly, the present invention provides a method of preventing,reducing or inhibiting fetal loss comprising administering an effectiveamount of a CD200 protein or a nucleic acid sequence encoding a CD200protein to an animal in need thereof. The invention includes a use of aneffective amount of a CD200 protein on a nucleic acid molecules encodinga CD200 protein to prevent or inhibit fetal loss. The CD200 protein maybe from any species and may be the full length sequence or a fragmentthereof that is capable of preventing or inhibiting fetal loss.

One of skill in the art can determine whether or not a particular CD200or fragment thereof is useful in preventing fetal loss. As mentionedabove, one of skill in the art can readily test a CD200 or CD200fragment for its ability to suppress an immune response using known invitro assays. In addition the CD200 or CD200 fragment can also be testedfor its ability to prevent fetal loss in an animal model. For example,one could use the model described in Example 14 wherein the ability ofCD200 to prevent cytokine induced abortion in abortion-prone CBA×DBA/2mice is assessed. Further, mice pre-immunized with anti-phospholipid mayalso be used.

(iii) Graft Rejection

In another embodiment, the present invention provides a method ofsuppressing an immune response to a transplanted organ, cell or tissuein a recipient animal comprising administering an effective amount of aCD200 protein or a nucleic acid sequence encoding a CD200 protein to therecipient animal, preferably prior to the transplantation of the organor tissue. The invention includes a use of an effective amount of aCD200 protein or a nucleic acid sequence encoding a CD200 protein tosuppress an immune response to a transplanted organ, cell or tissue.

The recipient can be any member of the animal kingdom including rodents,pigs, cats, dogs, ruminants, non-human primates and preferably humans.The organ, cell or tissue to be transplanted can be from the samespecies as the recipient (allograft) or can be from another species(xenograft). The tissues, cells or organs can be any tissue or organincluding heart, liver, kidney, lung, pancreas, pancreatic islets, braintissue, cornea, bone, intestine, skin and haematopoietic cells and stemcells.

One of skill in the art can determine whether or not a particular CD200or fragment thereof is useful in preventing graft rejection. Asmentioned above, one of skill in the art can readily test a CD200 orCD200 fragment for its ability to suppress an immune response usingknown in vitro assays. In addition the CD200 or CD200 fragment can alsobe tested for its ability to prevent graft rejection in an animal model.For example, one could use the animal models described in Examples 3 to6.

The method of the invention may be used to prevent graft versus hostdisease wherein the immune cells in the transplant mount an immuneattack on the recipient's immune system. This can occur when the tissueto be transplanted contains immune cells such as when bone marrow orlymphoid tissue is transplanted when treating leukemias, aplasticanemias and enzyme or immune deficiencies, for example.

Accordingly, in another embodiment, the present invention provides amethod of preventing or inhibiting graft versus host disease in arecipient animal receiving an organ or tissue transplant comprisingadministering an effective amount of a CD200 protein or a nucleic acidsequence encoding a CD200 protein to the organ or tissue prior to thetransplantation in the recipient animal. The invention includes a use ofan effective amount of a CD200 protein or a nucleic acid moleculeencoding a CD200 protein to prevent or inhibit graft versus hostdisease.

(iv) Allergy

As stated previously, the method of the present invention may also beused to treat or prevent an allergic reaction. In an allergic reaction,the immune system mounts an attack against a generally harmless,innocuous antigen or allergen. Allergies that may be prevented ortreated using the methods of the invention include, but are not limitedto, hay fever, asthma, atopic eczema as well as allergies to poison oakand ivy, house dust mites, bee pollen, nuts, shellfish, penicillin andnumerous others.

Accordingly, in a further embodiment, the present invention provides amethod of preventing or treating an allergy comprising administering aneffective amount of a CD200 protein or a nucleic acid sequence encodinga CD200 protein to an animal having or suspected of having an allergy.The invention includes a use of an effective amount of a CD200 proteinor a nucleic acid molecule encoding a CD200 protein to prevent or treatan allergy.

One of skill in the art can determine whether or not a particular CD200or fragment thereof is useful in preventing allergy. As mentioned above,one of skill in the art can readily test a CD200 or CD200 fragment forits ability to suppress an immune response using known in vitro assays.In addition the CD200 or CD200 fragment can also be tested for itsability to prevent allergy in an animal model.

2. Preventing Immune Suppression

In another aspect, the present invention provides a method of preventingimmune suppression or inducing or augmenting an immune responsecomprising administering an effective amount of an agent that inhibitsCD200 to an animal in need thereof.

There are a large number of situations whereby it is desirable toprevent immune suppression including, but not limited to, the treatmentof infections, cancer and Acquired Immune Deficiency Syndrome and theinduction of fetal loss. One may also wish to induce an autoimmunereaction to develop an animal model for treating or studying autoimmunedisease.

The agent that inhibits CD200 can be any agent that decreases theexpression or activity of a CD200 protein such that the immunesuppression caused by CD200 is reduced, inhibited and/or prevented. Theagent that inhibits CD200 can be any agent that decreases the expressionor activity of a CD200 protein such that the immune suppression causedby CD200 is reduced, inhibited and/or prevented. Such agents can beselected from agents that inhibit CD200 activity (such as antibodies,CD200 ligands, small molecules), agents that inhibit CD200 expression(such as antisense molecules) or agents that inhibit the interaction ofCD200 with its receptor (such as soluble CD200 receptor and antibodiesthat bind the CD200 receptor). Specific agents that may be used areprovided in Section B.

One of skill in the art can readily determine whether or not aparticular agent is effective in inhibiting CD200. For example, theagent can be tested in in vitro assays to determine if the function oractivity of CD200 is inhibited. The agent can also be tested for itsability to induce an immune response using in vitro immune assaysincluding, but not limited to, enhancing a cytotoxic T cell response;inducing interleukin-2 (IL-2) production; inducing IFNγ production;inducing a Th1 cytokine profile; inhibiting IL-4 production; inhibitingTGFβ production; inhibiting IL-10 production; inhibiting a Th2 cytokineprofile and any other assay that would be known to one of skill in theart to be useful in detecting immune activation.

(i) Fetal Loss

In one embodiment, the present invention provides a method of inducingfetal loss comprising administering an effective amount of an agent thatinhibits CD200 to an animal in need thereof. The animal is preferably apregnant female.

One of skill in the art can determine whether a particular agent isuseful in inducing fetal loss. As mentioned above, one can test theagent for its ability to induce an immune response using known in vitroassays. In addition, the agent can be tested in an animal model, forexample as described in Example 11, wherein the agent is administered toa pregnant rodent.

(ii) Tumor Growth

The inventors have shown that inhibiting the immune suppression causedby CD200 can be used to treat tumors. The data provided in Example 9demonstrates that increased expression of CD200 allows a tumor to evadethe immune system which results in tumor growth and increased mortality.However, administering antibodies to CD200 to animals having a tumor andincreased CD200 levels results in improved tumor immunity, reduced tumorcell growth and improved survival. This was demonstrated with twodifferent types of tumor, an EL4 tumor which is a thymoma and a C1498tumor which is a myeloid tumor. Therefore, inhibiting CD200 can be usedto treat cancers that are treatable by inhibiting or preventing immunesuppression caused by CD200.

In another embodiment, the present invention provides a method ofinhibiting, preventing or reducing tumor cell growth comprisingadministering an effective amount of an agent that inhibits CD200 to acell or an animal in need thereof. Preferably, the animal is an animalwith cancer, more preferably human.

One of skill in the art can determine whether a particular agent isuseful in inhibiting tumor cell growth. As mentioned above, one can testthe agent for its ability to induce an immune response using known invitro assays. In addition, the agent can be tested in an animal model,for example as described in Examples 9 and 10, wherein the agent isadministered to an animal with cancer.

The term “inhibiting or reducing tumor cell growth” means that the agentthat inhibits CD200 causes an inhibition or reduction in the growth ormetastasis of a tumor as compared to the growth observed in the absenceof the agent. The agent may also be used prophylactically to prevent thegrowth of tumor cells.

The tumor cell can be any type of cancer that is treatable by inhibitingCD200 including, but not limited to, hematopoietic cell cancers(including leukemias and lymphomas), colon cancer, lung cancer, kidneycancer, pancreas cancer, endometrial cancer, thyroid cancer, oralcancer, laryngeal cancer, hepatocellular cancer, bile duct cancer,squamous cell carcinoma, prostate cancer, breast cancer, cervicalcancer, colorectal cancer, melanomas. and any other tumors which areantigenic or weakly antigenic. This could include, for example,EBV-induced neoplasms, and neoplasms occurring in immunosuppressedpateints, e.g. transplant patients, AIDS patients, etc.

Once a particular tumor has been identified for treatment, one of skillin the art could determine whether or not it can evade the immune systemthrough the CD200 pathway. In particular, one can immunize an animalwith the tumor and determine whether or not it increases CD200expression as was done for the tumor cells used in Example 9. Further,one can also directly assess the tumor to see if it expresses CD200using methods that are routine in the art.

B. CD200 Inhibitors

In the above therapeutic methods for preventing immune suppression (oraugmenting an immune response), any agent that can inhibit CD200 may beused, some of which are discussed below.

(a) Antibodies

In a preferred embodiment, the agent that inhibits CD200 is a CD200specific antibody. The present inventor has prepared antibodies to CD200which are described in Examples 4 and 5. Antibodies to CD200 may also beprepared using techniques known in the art such as those described byKohler and Milstein, Nature 256, 495 (1975) and in U.S. Pat. Nos. RE32,011; 4,902,614; 4,543,439; and 4,411,993, which are incorporatedherein by reference. (See also Monoclonal Antibodies, Hybridomas: A NewDimension in Biological Analyses, Plenum Press, Kennett, McKearn, andBechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow andLane (eds.), Cold Spring Harbor Laboratory Press, 1988, which are alsoincorporated herein by reference). Within the context of the presentinvention, antibodies are understood to include monoclonal antibodies,polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab′)₂) andrecombinantly produced binding partners.

Conventional methods can be used to prepare the antibodies. For example,by using the CD200 protein, polyclonal antisera or monoclonal antibodiescan be made using standard methods. A mammal, (e.g., a mouse, hamster,or rabbit) can be immunized with an immunogenic form of the CD200protein which elicits an antibody response in the mammal. Techniques forconferring immunogenicity on a peptide include conjugation to carriersor other techniques well known in the art. For example, the peptide canbe administered in the presence of adjuvant. The progress ofimmunization can be monitored by detection of antibody titers in plasmaor serum. Standard ELISA or other immunoassay procedures can be usedwith the immunogen as antigen to assess the levels of antibodies.Following immunization, antisera can be obtained and, if desired,polyclonal antibodies isolated from the sera.

To produce monoclonal antibodies, antibody producing cells (lymphocytes)can be harvested from an immunized animal and fused with myeloma cellsby standard somatic cell fusion procedures thus immortalizing thesecells and yielding hybridoma cells. Such techniques are well known inthe art, (e.g., the hybridoma technique originally developed by Kohlerand Milstein (Nature 256, 495-497 (1975)) as well as other techniquessuch as the human B-cell hybridoma technique (Kozbor et al., Immunol.Today 4, 72 (1983)); the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al. Monoclonal Antibodies in CancerTherapy (1985) Allen R. Bliss, Inc., pages 77-96); and screening ofcombinatorial antibody libraries (Huse et al., Science 246, 1275(1989)). Hybridoma cells can be screened immunochemically for productionof antibodies specifically reactive with the CD200 protein and themonoclonal antibodies can be isolated. Therefore, the invention alsocontemplates hybridoma cells secreting monoclonal antibodies withspecificity for CD200.

The term “antibody” as used herein is intended to include fragmentsthereof which also specifically react with CD200 or a peptide thereof.Antibodies can be fragmented using conventional techniques and thefragments screened for utility in the same manner as described above.For example, F(ab′)₂ fragments can be generated by treating antibodywith pepsin. The resulting F(ab′)2 fragment can be treated to reducedisulfide bridges to produce Fab′ fragments.

Chimeric antibody derivatives, i.e., antibody molecules that combine anon-human animal variable region and a human constant region are alsocontemplated within the scope of the invention. Chimeric antibodymolecules can include, for example, the antigen binding domain from anantibody of a mouse, rat, or other species, with human constant regions.Conventional methods may be used to make chimeric antibodies containingthe immunoglobulin variable region which recognizes a CD200 protein(See, for example, Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 81,6851 (1985); Takeda et al., Nature 314, 452 (1985), Cabilly et al., U.S.Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi etal., European Patent Publication EP171496; European Patent Publication0173494, United Kingdom patent GB 2177096B).

Monoclonal or chimeric antibodies specifically reactive with the CD200as described herein can be further humanized by producing human constantregion chimeras, in which parts of the variable regions, particularlythe conserved framework regions of the antigen-binding domain, are ofhuman origin and only the hypervariable regions are of non human origin.Such immunoglobulin molecules may be made by techniques known in the art(e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80, 7308-7312 (1983);Kozbor et al., Immunology Today, 4, 7279 (1983); Olsson et al., Meth.Enzymol., 92, 3-16 (1982); and PCT Publication WO 92/06193 or EP0239400). Humanized antibodies can also be commercially produced(Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain.)

Specific antibodies, or antibody fragments reactive against CD200 mayalso be generated by screening expression libraries encodingimmunoglobulin genes, or portions thereof, expressed in bacteria withpeptides produced from nucleic acid molecules of the present invention.For example, complete Fab fragments, VH regions and FV regions can beexpressed in bacteria using phage expression libraries (See for exampleWard et al., Nature 341, 544-546: (1989); Huse et al., Science 246,1275-1281 (1989); and McCafferty et al. Nature 348, 552-554 (1990)).

Accordingly, the present invention provides a method of preventingimmune suppression or inhibiting, preventing or reducing tumor cellgrowth comprising administering an effective amount of an antibody thatinhibits CD200 to an animal in need thereof. The invention also includesthe use of an antibody that inhibits CD200 to prepare a medicament toinhibit, prevent or reduce tumor cell growth.

The present invention further provides a method of inducing fetal losscomprising administering an effective amount of an antibody thatinhibits CD200 to an animal in need thereof.

(b) Antisense Oligonucleotides

In another embodiment, the CD200 inhibitor is an antisenseoligonucleotide that inhibits the expression of CD200. Antisenseoligonucleotides that are complimentary to a nucleic acid sequence froma CD200 gene can be used in the methods of the present invention toinhibit CD200. The present inventor has prepared antisenseoligonucleotides to CD200 which are described in Example 3.

Accordingly, the present invention provides a method of preventingimmune suppression comprising administering an effective amount of anantisense oligonucleotide that is complimentary to a nucleic acidsequence from a CD200 gene to an animal in need thereof.

In another embodiment, the present invention provides a method ofinducing fetal loss or inhibiting tumor cell growth comprisingadministering an effective amount of an antisense oligonucleotide thatis complimentary to a nucleic acid sequence from a CD200 gene to ananimal in need thereof.

The term antisense oligonucleotide as used herein means a nucleotidesequence that is complimentary to its target.

In one embodiment of the invention, the present invention provides anantisense oligonucleotide that is complimentary to a nucleic acidmolecule having a sequence as shown in FIG. 7 (SEQ ID Nos:18, 20, 22),wherein T can also be U, or a fragment thereof.

The term “oligonucleotide” refers to an oligomer or polymer ofnucleotide or nucleoside monomers consisting of naturally occurringbases, sugars, and intersugar (backbone) linkages. The term alsoincludes modified or substituted oligomers comprising non-naturallyoccurring monomers or portions thereof, which function similarly. Suchmodified or substituted oligonucleotides may be preferred over naturallyoccurring forms because of properties such as enhanced cellular uptake,or increased stability in the presence of nucleases. The term alsoincludes chimeric oligonucleotides which contain two or more chemicallydistinct regions. For example, chimeric oligonucleotides may contain atleast one region of modified nucleotides that confer beneficialproperties (e.g. increased nuclease resistance, increased uptake intocells), or two or more oligonucleotides of the invention may be joinedto form a chimeric oligonucleotide.

The antisense oligonucleotides of the present invention may beribonucleic or deoxyribonucleic acids and may contain naturallyoccurring bases including adenine, guanine, cytosine, thymidine anduracil. The oligonucleotides may also contain modified bases such asxanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and otheralkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-azacytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine,8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyladenine and other 8-substituted adenines, 8-halo guanines, 8-aminoguanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine andother 8-substituted guanines, other aza and deaza uracils, thymidines,cytosines, adenines, or guanines, 5-trifluoromethyl uracil and5-trifluoro cytosine.

Other antisense oligonucleotides of the invention may contain modifiedphosphorous, oxygen heteroatoms in the phosphate backbone, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. For example, the antisenseoligonucleotides may contain phosphorothioates, phosphotriesters, methylphosphonates, and phosphorodithioates. In an embodiment of the inventionthere are phosphorothioate bonds links between the four to six3′-terminus bases. In another embodiment phosphorothioate bonds link allthe nucleotides.

The antisense oligonucleotides of the invention may also comprisenucleotide analogs that may be better suited as therapeutic orexperimental reagents. An example of an oligonucleotide analogue is apeptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphatebackbone in the DNA (or RNA), is replaced with a polyamide backbonewhich is similar to that found in peptides (P. E. Nielsen, et al Science1991, 254, 1497). PNA analogues have been shown to be resistant todegradation by enzymes and to have extended lives in vivo and in vitro.PNAs also bind stronger to a complimentary DNA sequence due to the lackof charge repulsion between the PNA strand and the DNA strand. Otheroligonucleotides may contain nucleotides containing polymer backbones,cyclic backbones, or acyclic backbones. For example, the nucleotides mayhave morpholino backbone structures (U.S. Pat. No. 5,034,506).Oligonucleotides may also contain groups such as reporter groups, agroup for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an antisense oligonucleotide. Antisense oligonucleotides may alsohave sugar mimetics.

The antisense nucleic acid molecules may be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. The antisense nucleic acid molecules of the invention or a fragmentthereof, may be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed with mRNA or the native gene e.g.phosphorothioate derivatives and acridine substituted nucleotides. Theantisense sequences may be produced biologically using an expressionvector introduced into cells in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense sequences are producedunder the control of a high efficiency regulatory region, the activityof which may be determined by the cell type into which the vector isintroduced.

(c) Other CD200 Inhibitors

In addition to antibodies and antisense molecules, other agents thatinhibit CD200 may also be used in the present invention.

Accordingly, the present invention also includes the isolation of otherligands or molecules that can bind to CD200 or the CD200 receptor.Biological samples and commercially available libraries may be testedfor proteins that bind to CD200 or the CD200 receptor. In addition,antibodies prepared to the CD200 or the CD200 receptor may be used toisolate other peptides with CD200 or CD200 receptor binding affinity.For example, labelled antibodies may be used to probe phage displayslibraries or biological samples.

Conditions which permit the formation of protein complexes may beselected having regard to factors such as the nature and amounts of thesubstance and the protein.

The substance-protein complex, free substance or non-complexed proteinsmay be isolated by conventional isolation techniques, for example,salting out, chromatography, electrophoresis, gel filtration,fractionation, absorption, polyacrylamide gel electrophoresis,agglutination, or combinations thereof. To facilitate the assay of thecomponents, the antibodies, proteins, or substances may be labelled witha detectable substance.

Once potential binding partners have been isolated, screening methodsmay be designed in order to determine if the molecules that bind to theCD200 peptide or CD200 receptor and are useful in the methods of thepresent invention.

Therefore, the invention also provides methods for identifyingsubstances which are capable of binding to the CD200. In particular, themethods may be used to identify substances which are capable of bindingto and which suppress the effects of CD200. Accordingly the inventionprovides a method of identifying substances which bind with CD200,comprising the steps of:

(a) reacting CD200 and a substance, under conditions which allow forformation of a complex, and

(b) assaying for complexes, for free substance, and for non complexedCD200.

Substances which can bind with the CD200 of the invention may beidentified by reacting CD200 with a substance which potentially binds tothe CD200, and assaying for complexes, for free substance, or fornon-complexed CD200. Any assay system or testing method that detectsprotein-protein interactions may be used includingco-immunoprecipitation, crosslinking and co-purification throughgradients or chromatographic columns may be used. Additionally, x-raycrystallographic studies may be used as a means of evaluatinginteractions with substances and molecules. For example, purifiedrecombinant molecules in a complex of the invention when crystallized ina suitable form are amenable to detection of intra-molecularinteractions by x-ray crystallography. Spectroscopy may also be used todetect interactions and in particular, Q-TOF instrumentation may beused. Biological samples and commercially available libraries may betested for CD200-binding peptides. In addition, antibodies prepared tothe peptides of the invention may be used to isolate other peptides withCD200 binding affinity. For example, labelled antibodies may be used toprobe phage display libraries or biological samples. In this respectpeptides of the invention may be developed using a biological expressionsystem. The use of these systems allows the production of largelibraries of random peptide sequences and the screening of theselibraries for peptide sequences that bind to particular proteins.Libraries may be produced by cloning synthetic DNA that encodes randompeptide sequences into appropriate expression vectors. (see Christian etal. 1992, J. Mol. Biol. 227:711; Devlin et al., 1990 Science 249:404;Cwirla et al. 1990, Proc. Natl. Acad, Sci. USA, 87:6378). Libraries mayalso be constructed by concurrent synthesis of overlapping peptides (seeU.S. Pat. No. 4,708,871).

It will be understood that the agonist and antagonist that can beassayed using the methods of the invention may act on one or more of thebinding sites on the protein or substance including agonist bindingsites, competitive antagonist binding sites, non-competitive antagonistbinding sites or allosteric sites.

The invention also makes it possible to screen for antagonists thatinhibit the effects of an agonist of the interaction of CD200 with asubstance which is capable of binding to CD200. Thus, the invention maybe used to assay for a substance that competes for the same binding siteof CD200. As such it will also be appreciated that intracellularsubstances which are capable of binding to CD200 may be identified usingthe methods described herein.

The reagents suitable for applying the methods of the invention toevaluate substances and compounds that affect or modulate a CD200 may bepackaged into convenient kits providing the necessary materials packagedinto suitable containers. The kits may also include suitable supportsuseful in performing the methods of the invention.

C. Compositions

The invention also includes pharmaceutical compositions containing CD200proteins or nucleic acids for use in immune suppression as well aspharmaceutical compositions containing a CD200 inhibitor for use inpreventing immune suppression.

Such pharmaceutical compositions can be for intralesional, intravenous,topical, rectal, parenteral, local, inhalant or subcutaneous,intradermal, intramuscular, intrathecal, transperitoneal, oral, andintracerebral use. The composition can be in liquid, solid or semisolidform, for example pills, tablets, creams, gelatin capsules, capsules,suppositories, soft gelatin capsules, gels, membranes, tubelets,solutions or suspensions.

The pharmaceutical compositions of the invention can be intended foradministration to humans or animals. Dosages to be administered dependon individual needs, on the desired effect and on the chosen route ofadministration.

The pharmaceutical compositions can be prepared by per se known methodsfor the preparation of pharmaceutically acceptable compositions whichcan be administered to patients, and such that an effective quantity ofthe active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA 1985).

On this basis, the pharmaceutical compositions include, albeit notexclusively, the active compound or substance in association with one ormore pharmaceutically acceptable vehicles or diluents, and contained inbuffered solutions with a suitable pH and iso-osmotic with thephysiological fluids. The pharmaceutical compositions may additionallycontain other agents such as immunosuppressive drugs or antibodies toenhance immune tolerance or immunostimulatory agents to enhance theimmune response.

In one embodiment, the pharmaceutical composition for use in immunesuppression, treating autoimmunity, preventing graft rejection orpreventing fetal loss comprises an effective amount of a CD200 proteinin admixture with a pharmaceutically acceptable diluent or carrier. TheCD200 protein is preferably prepared as an immunoadhesion molecule insoluble form which can be administered to the patient.

In another embodiment, the pharmaceutical composition for use in immunesuppression, treating autoimmunity, preventing graft rejection orpreventing fetal loss comprises an effective amount of a nucleic acidmolecule encoding a CD200 protein in admixture with a pharmaceuticallyacceptable diluent or carrier.

The nucleic acid molecules of the invention encoding a CD200 protein maybe used in gene therapy to induce immune tolerance. Recombinantmolecules comprising a nucleic acid sequence encoding a CD200 protein,or fragment thereof, may be directly introduced into cells or tissues invivo using delivery vehicles such as retroviral vectors, adenoviralvectors and DNA virus vectors. They may also be introduced into cells invivo using physical techniques such as microinjection andelectroporation or chemical methods such as coprecipitation andincorporation of DNA into liposomes. Recombinant molecules may also bedelivered in the form of an aerosol or by lavage. The nucleic acidmolecules of the invention may also be applied extracellularly such asby direct injection into cells. The nucleic acid molecules encodingCD200 are preferably prepared as a fusion with a nucleic acid moleculeencoding an immunoglobulin (Ig) Fc region. As such, the CD200 proteinwill be expressed in vivo as a soluble fusion protein.

In another aspect, the pharmaceutical composition for use in preventingimmune suppression, inhibiting tumor growth or inducing fetal losscomprises an effective amount of a CD200 inhibitor in admixture with apharmaceutically acceptable diluent or carrier. Such compositions may beadministered as a vaccine either alone or in combination with otheractive agents.

In one embodiment, the pharmaceutical composition for use in preventingimmune suppression, inhibiting tumor growth or inducing fetal losscomprises an effective amount of an antibody to CD200 in admixture witha pharmaceutically acceptable diluent or carrier. The antibodies may bedelivered intravenously.

In another embodiment, the pharmaceutical composition for use inpreventing immune suppression, inhibiting tumor growth or inducing fetalloss comprises an effective amount of an antisense oligonucleotidenucleic acid complimentary to a nucleic acid sequence from a CD200 genein admixture with a pharmaceutically acceptable diluent or carrier. Theoligonucleotide molecules may be administered as described above for thecompositions containing CD200 nucleic acid sequences.

D. Murine CD200

The inventor has cloned and sequenced the murine CD200 gene.Accordingly, the invention also includes an isolated nucleic acidsequence encoding a murine CD200 gene and having the sequence shown inFIG. 7 and SEQ ID NO:22.

The term “isolated” refers to a nucleic acid substantially free ofcellular material or culture medium when produced by recombinant DNAtechniques, or chemical precursors, or other chemicals when chemicallysynthesized. The term “nucleic acid” is intended to include DNA and RNAand can be either double stranded or single stranded.

Preferably, the purified and isolated nucleic acid molecule of theinvention comprises (a) a nucleic acid sequence as shown in SEQ IDNO:22, wherein T can also be U; (b) nucleic acid sequences complementaryto (a); (c) a fragment of (a) or (b) that is at least 15 bases,preferably 20 to 30 bases, and which will hybridize to (a) or (b) understringent hybridization conditions; or (a) a nucleic acid moleculediffering from any of the nucleic acids of (a) or (b) in codon sequencesdue to the degeneracy of the genetic code.

It will be appreciated that the invention includes nucleic acidmolecules encoding truncations of the murine CD200 proteins of theinvention, and analogs and homologs of the proteins of the invention andtruncations thereof, as described below. It will further be appreciatedthat variant forms of the nucleic acid molecules of the invention whicharise by alternative splicing of an mRNA corresponding to a cDNA of theinvention are encompassed by the invention.

An isolated nucleic acid molecule of the invention which is DNA can alsobe isolated by selectively amplifying a nucleic acid encoding a novelprotein of the invention using the polymerase chain reaction (PCR)methods and cDNA or genomic DNA. It is possible to design syntheticoligonucleotide primers from the nucleic acid molecules as shown in FIG.7 and SEQ ID NO:22 for use in PCR. A nucleic acid can be amplified fromcDNA or genomic DNA using these oligonucleotide primers and standard PCRamplification techniques. The nucleic acid so amplified can be clonedinto an appropriate vector and characterized by DNA sequence analysis.It will be appreciated that cDNA may be prepared from mRNA, by isolatingtotal cellular mRNA by a variety of techniques, for example, by usingthe guanidinium-thiocyanate extraction procedure of Chirgwin et al.,Biochemistry, 18, 5294-5299 (1979). cDNA is then synthesized from themRNA using reverse transcriptase (for example, Moloney MLV reversetranscriptase available from Gibco/BRL, Bethesda, Md., or AMV reversetranscriptase available from Seikagaku America, Inc., St. Petersburg,Fla.).

An isolated nucleic acid molecule of the invention which is RNA can beisolated by cloning a cDNA encoding a novel protein of the inventioninto an appropriate vector which allows for transcription of the cDNA toproduce an RNA molecule which encodes a CD200 protein of the invention.For example, a cDNA can be cloned downstream of a bacteriophagepromoter, (e.g. a T7 promoter) in a vector, cDNA can be transcribed invitro with T7 polymerase, and the resultant RNA can be isolated bystandard techniques.

A nucleic acid molecule of the invention may also be chemicallysynthesized using standard techniques. Various methods of chemicallysynthesizing polydeoxynucleotides are known, including solid-phasesynthesis which, like peptide synthesis, has been fully automated incommercially available DNA synthesizers (See e.g., Itakura et al. U.S.Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; andItakura U.S. Pat. Nos. 4,401,796 and 4,373,071).

The sequence of a nucleic acid molecule of the invention may be invertedrelative to its normal presentation for transcription to produce anantisense nucleic acid molecule. Preferably, an antisense sequence isconstructed by inverting a region preceding the initiation codon or anunconserved region. In particular, the nucleic acid sequences containedin the nucleic acid molecules of the invention or a fragment thereof,preferably a nucleic acid sequence shown in FIG. 7 and SEQ ID NO:22 maybe inverted relative to its normal presentation for transcription toproduce antisense nucleic acid molecules.

The antisense nucleic acid molecules of the invention or a fragmentthereof, may be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed with mRNA or the native gene e.g.phosphorothioate derivatives and acridine substituted nucleotides. Theantisense sequences may be produced biologically using an expressionvector introduced into cells in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense sequences are producedunder the control of a high efficiency regulatory region, the activityof which may be determined by the cell type into which the vector isintroduced.

The invention also provides nucleic acids encoding fusion proteinscomprising a CD200 protein of the invention and a selected protein, or aselectable marker protein.

The invention further includes an isolated protein which has the aminoacid sequence as shown in FIG. 8 and SEQ ID NO:2.

Within the context of the present invention, a protein of the inventionmay include various structural forms of the primary protein which retainbiological activity. For example, a protein of the invention may be inthe form of acidic or basic salts or in neutral form. In addition,individual amino acid residues may be modified by oxidation orreduction.

In addition to the full length amino acid sequence (FIG. 8), the proteinof the present invention may also include truncations of the protein,and analogs, and homologs of the protein and truncations thereof asdescribed herein. Truncated proteins may comprise peptides of at leastfifteen amino acid residues.

Analogs of the protein having the amino acid sequence shown in FIG. 8,and/or truncations thereof as described herein, may include, but are notlimited to an amino acid sequence containing one or more amino acidsubstitutions, insertions, and/or deletions. Amino acid substitutionsmay be of a conserved or non-conserved nature. Conserved amino acidsubstitutions involve replacing one or more amino acids of the proteinsof the invention with amino acids of similar charge, size, and/orhydrophobicity characteristics. When only conserved substitutions aremade the resulting analog should be functionally equivalent.Non-conserved substitutions involve replacing one or more amino acids ofthe amino acid sequence with one or more amino acids which possessdissimilar charge, size, and/or hydrophobicity characteristics.

One or more amino acid insertions may be introduced into the amino acidsequences shown in FIG. 8. Amino acid insertions may consist of singleamino acid residues or sequential amino acids ranging from 2 to 15 aminoacids in length. For example, amino acid insertions may be used torender the protein is no longer active. This procedure may be used invivo to inhibit the activity of a protein of the invention.

Deletions may consist of the removal of one or more amino acids, ordiscrete portions from the amino acid sequence shown in FIG. 8. Thedeleted amino acids may or may not be contiguous. The lower limit lengthof the resulting analog with a deletion mutation is about 10 aminoacids, preferably 100 amino acids.

Analogs of a protein of the invention may be prepared by introducingmutations in the nucleotide sequence encoding the protein. Mutations innucleotide sequences constructed for expression of analogs of a proteinof the invention must preserve the reading frame of the codingsequences. Furthermore, the mutations will preferably not createcomplementary regions that could hybridize to produce secondary mRNAstructures, such as loops or hairpins, which could adversely affecttranslation of the receptor mRNA.

Mutations may be introduced at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site specific mutagenesisprocedures may be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Deletion or truncation of a protein of the invention may alsobe constructed by utilizing convenient restriction endonuclease sitesadjacent to the desired deletion. Subsequent to restriction, overhangsmay be filled in, and the DNA religated. Exemplary methods of making thealterations set forth above are disclosed by Sambrook et al (MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, 1989).

The invention also contemplates isoforms of the proteins of theinvention. An isoform contains the same number and kinds of amino acidsas a protein of the invention, but the isoform has a different molecularstructure. The isoforms contemplated by the present invention are thosehaving the same properties as a protein of the invention as describedherein.

The present invention also includes a protein of the inventionconjugated with a selected protein, or a selectable marker protein toproduce fusion proteins. Additionally, immunogenic portions of a proteinof the invention are within the scope of the invention.

The proteins of the invention (including truncations, analogs, etc.) maybe prepared using recombinant DNA methods. Accordingly, the nucleic acidmolecules of the present invention having a sequence which encodes aprotein of the invention may be incorporated in a known manner into anappropriate expression vector which ensures good expression of theprotein. Possible expression vectors include but are not limited tocosmids, plasmids, or modified viruses (e.g. replication defectiveretroviruses, adenoviruses and adeno-associated viruses), so long as thevector is compatible with the host cell used. The expression vectors are“suitable for transformation of a host cell”, means that the expressionvectors contain a nucleic acid molecule of the invention and regulatorysequences selected on the basis of the host cells to be used forexpression, which is operatively linked to the nucleic acid molecule.Operatively linked is intended to mean that the nucleic acid is linkedto regulatory sequences in a manner which allows expression of thenucleic acid.

The invention therefore contemplates a recombinant expression vector ofthe invention containing a nucleic acid molecule of the invention, or afragment thereof, and the necessary regulatory sequences for thetranscription and translation of the inserted protein-sequence. Suchexpression vectors may be useful in the above-described therapies usinga nucleic acid sequence encoding a CD200 protein. Suitable regulatorysequences may be derived from a variety of sources, including bacterial,fungal, or viral genes (For example, see the regulatory sequencesdescribed in Goeddel, Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). Selection of appropriateregulatory sequences is dependent on the host cell chosen, and may bereadily accomplished by one of ordinary skill in the art. Examples ofsuch regulatory sequences include: a transcriptional promoter andenhancer or RNA polymerase binding sequence, a ribosomal bindingsequence, including a translation initiation signal. Additionally,depending on the host cell chosen and the vector employed, othersequences, such as an origin of replication, additional DNA restrictionsites, enhancers, and sequences conferring inducibility of transcriptionmay be incorporated into the expression vector. It will also beappreciated that the necessary regulatory sequences may be supplied bythe native protein and/or its flanking regions.

The invention further provides a recombinant expression vectorcomprising a DNA nucleic acid molecule of the invention cloned into theexpression vector in an antisense orientation. That is, the DNA moleculeis operatively linked to a regulatory sequence in a manner which allowsfor expression, by transcription of the DNA molecule, of an RNA moleculewhich is antisense to a nucleotide sequence comprising the nucleotidesas shown in FIG. 7 and SEQ ID NO:22. Regulatory sequences operativelylinked to the antisense nucleic acid can be chosen which direct thecontinuous expression of the antisense RNA molecule.

The recombinant expression vectors of the invention may also contain aselectable marker gene which facilitates the selection of host cellstransformed or transfected with a recombinant molecule of the invention.Examples of selectable marker genes are genes encoding a protein such asG418 and hygromycin which confer resistance to certain drugs,β-galactosidase, chloramphenicol acetyltransferase, or fireflyluciferase. Transcription of the selectable marker gene is monitored bychanges in the concentration of the selectable marker protein such asb-galactosidase, chloramphenicol acetyltransferase, or fireflyluciferase. If the selectable marker gene encodes a protein conferringantibiotic resistance such as neomycin resistance transformant cells canbe selected with G418. Cells that have incorporated the selectablemarker gene will survive, while the other cells die. This makes itpossible to visualize and assay for expression of recombinant expressionvectors of the invention and in particular to determine the effect of amutation on expression and phenotype. It will be appreciated thatselectable markers can be introduced on a separate vector from thenucleic acid of interest.

The recombinant expression vectors may also contain genes which encode afusion moiety which provides increased expression of the recombinantprotein; increased solubility of the recombinant protein; and aid in thepurification of a target recombinant protein by acting as a ligand inaffinity purification. For example, a proteolytic cleavage site may beadded to the target recombinant protein to allow separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein.

Recombinant expression vectors can be introduced into host cells toproduce a transformant host cell. The term “transformant host cell” isintended to include prokaryotic and eukaryotic cells which have beentransformed or transfected with a recombinant expression vector of theinvention. The terms “transformed with”, “transfected with”,“transformation” and “transfection” are intended to encompassintroduction of nucleic acid (e.g. a vector) into a cell by one of manypossible techniques known in the art. Prokaryotic cells can betransformed with nucleic acid by, for example, electroporation orcalcium-chloride mediated transformation. Nucleic acid can be introducedinto mammalian cells via conventional techniques such as calciumphosphate or calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofectin, electroporation or microinjection. Suitablemethods for transforming and transfecting host cells can be found inSambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition,Cold Spring Harbor Laboratory press (1989)), and other laboratorytextbooks.

Suitable host cells include a wide variety of prokaryotic and eukaryotichost cells. For example, the proteins of the invention may be expressedin bacterial cells such as E. coli, insect cells (using baculovirus),yeast cells or mammalian cells. Other suitable host cells can be foundin Goeddel, Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1991).

The proteins of the invention may also be prepared by chemical synthesisusing techniques well known in the chemistry of proteins such as solidphase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) orsynthesis in homogenous solution (Houbenweyl, 1987, Methods of OrganicChemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart).

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES Example 1

This example demonstrates the increased expression of certain genesfollowing pv immunization.

Mice:

C3H/HEJ and C57BL/6 mice were purchased from The Jackson Laboratory, BarHarbor, Me. Mice were housed five/cage and allowed food and water adlibitum. All mice were used at 8-12 weeks of age.

Monoclonal Antibodies:

The following monoclonal antibodies (Mabs) from Pharmingen (San Diego,Calif.) were used: anti-IL-2 (JES6-1A12; biotinylated JES6-5H4);anti-IL-4 (11B11; biotinylated BVD6-24G2); anti-IFNγ (R4-6A2;biotinylated XMG1.2); anti-IL-10 (JES5-2A5; biotinylated SXC-1,Pharmingen, San Diego, Calif.); mouse IgG1 isotype control (clone 107.3,BALB/c anti-TNP). Strepavidin horse radish peroxidase and recombinantmouse GM-CSF was also purchased from Pharmingen (San Diego, Calif.).

NLDC-145 (anti-mouse dendritic cells), and F(ab′)₂ rabbit anti-rat IgGFITC conjugate (non-cross reactive with mouse IgG), or F(ab′)₂ rabbitanti-mouse IgG PE was obtained from Serotec, Canada.

Rabbit complement, L3T4, anti-thy1.2, anti-Ly2.2, anti-Ly2.1 (mouseIgG3), FITC-MAC-1 and mouse IgG1 anti-rat CD200 were obtained fromCedarlane Labs, Hornby, Ontario.

Anti-CD28 (PV-1) and anti-CTLA (UC10-4F10-11) were obtained from Drs. C.June and J. Bluestone respectively, while anti-B7-1, anti-B7-2 wereobtained from Dr. G. Powers. High titres of all 4 of the latterantibodies were produced by in vitro culture in a CELLMAX system (CELLCOInc., Germantown, Md., USA).

Preparation of Cells:

Spleen, Peyer's Patch (PP) and mesenteric lymph node (MLN) cellsuspensions were prepared aseptically from individual mice of thedifferent treated groups in each experiment.

Where dendritic cells were obtained by culture of bone marrow cells invitro the following technique was used (Gorczynski et al., 1996a). Bonemarrow plugs were aspirated from the femurs of donor male C57BL/6 (orBALB/c) mice, washed and resuspended in aF10. Cells were treatedsequentially with a mixture of antibodies (L3T4, anti-thy1.2,anti-Ly2.2) and rabbit complement and dead cells removed bycentrifugation over mouse lymphopaque (Cedarlane Labs, Ontario). Cellswere washed ×3 in aF10, and cultured in 10 ml aF10 in tissue cultureflasks, at a concentration of 2×10⁶/ml with 500 U/ml recombinant murineGM-CSF (Pharmingen, USA). Fresh GM-CSF was added at 36 hr intervals.Cells were separated over lymphopaque on days 3.5 and 7 of culture,again reculturing in aF10 with recombinant GM-CSF. At 10 days an aliquotof the sample was stained with NLDC-145 and FITC anti-rat IgG,anti-CD200 and PE anti-mouse IgG, FITC-anti-B7-1 or FITC anti-B7-2. Meanstaining with these antibodies using cells harvested from such cultureshas been 93%±7%, 14%±5%, 78%±9% and 27%±6% respectively. Remaining cellswere washed, and injected into the portal vein as described.

Portal Vein Immunizations and Renal Transplantation:

The pv immunizations and renal transplantation were performed asdescribed earlier (Gorczynski et al., 1994). All C3H mice received pv/ivimmunization with 15×10⁶ C57BL/6 10-day cultured, bone marrow derived,dendritic cells, followed by C57BL/6 kidney transplantation. Animalsreceived 1 intramoscular (im) injection with 10 mg/Kg cyclosporin A onthe day of transplantation. Mice were sacrificed for tissue harvest andRNA preparation 5 days after transplantation. In other studies animalswere sacrificed as described in the text.

Where monoclonal antibodies were injected into transplanted mice,animals received 100 mg intravenous (iv) at 2 day intervals (×4injections) beginning within 2 hours of transplantation.

Cytokine Production from Spleen Cells of Transplanted Mice:

In cultures used to assess induction of cytokine production spleenresponder cells stimulated with irradiated (2000R) C57BL/6 spleenstimulator cells in triplicate in aF10 have been used. In multiplestudies significant quantitative or qualitative differences in cytokineproduction from spleen, lymph node or Peyer's Patch of transplanted micehave not been seen. (Gorczynski et al., 1994b). Supernatants were pooledat 40 hr from replicate wells and assayed in triplicate in ELISA assaysfor lymphokine production. All capture antibodies, biotinylateddetection antibodies, and recombinant cytokines were obtained fromPharmingen (San Diego, Calif.—see above).

For IFNγ the assay used flat-bottomed 96-well Nunc plates (Gibco, BRL)coated with 100 ng/ml R4-6A2. Varying volumes of supernatant were boundin triplicate at 4° C., washed ×3, and biotinylated anti-IFNγ (XMG1.2)added. After washing, plates were incubated with strepavidin-horseradish peroxidase (Cedarlane Labs), developed with appropriate substrateand OD₄₀₅ determined using an ELISA plate reader. IL-10 was assayedusing a similar ELISA system with JES5-2A5 as the capture antibody, andbiotinylated SXC-1 as developing antibody. Each assay reliably detectedcytokine levels in the range 0.01 to 0.1 ng/ml. ELISA assays for IL-2and IL-4 used JES6-1A12 and 11B11 as capture antibodies, withbiotinylated JES6-5H4 or BVD6-24G2 as developing antibodies. Sensitivityof detection was 10 pg/ml for each cytokine.

Oligonucleotide Primers:

The primers used for PCR amplification for b-actin, and differentcytokines, are described in previous publications (Gorczynski, R. M.,1995a; Gorczynski, R. M., 1995b; Gorczynski, R. M., 1996a). In addition,the following oligonucleotides were synthesized.

cDNA synthesis primer for driver ds cDNA (DP): (SEQ ID NO: 3)5′-TTTTGTACAAGCTT₃₀-3′ Adapter 1 (Ad 1): (SEQ ID NO: 4)5′-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3′ Adapter 2 (Ad2):(SEQ ID NO: 5) 5′-TGTAGCGTGAAGACGACAGAAAGGGCGTGGTGCGGAGGGCGGT-3′PCR Primer1 (P1): (SEQ ID NO: 6) 5′-CTAATACGACTCACTATAGGGC-3′Nested Primer 1 (NP1): (SEQ ID NO: 7) 5′-TCGAGCGGCCGCCCGGGCAGGT-3′PCR Primer2 (P2): (SEQ ID NO: 8) 5′-TGTAGCGTGAAGACGACAGAA-3′Nested Primer 2 (NP2): (SEQ ID NO: 9) 5′-AGGGCGTGGTGCGGAGGGCGGT-3′Driver and Tester Preparation:

RNA was extracted from pooled mesenteric lymph node (MLN) and Peyer'sPatches (PP) of 5/group renal transplant mice with iv or pvimmunization. Poly(A)⁺ mRNA was prepared from the driver (iv) group, and2 mg material used for ds cDNA synthesis with 1 ng DP primer and a cDNASynthesis Kit (Clontech) with T4 DNA polymerase. The final cDNApreparation was digested with RsaI in a 50 ml reaction mixture with 15units enzyme (GIBCO) for 3 hrs, and the cDNA phenol-extracted, ethanolprecipitated, and resuspended in 7 ml of deionized water (concentrationapproximately 300 ng/ml).

RsaI digested ds tester cDNA (pv group) was prepared in a similarfashion. 50 ng of tester cDNA diluted in TE buffer was ligated with 2 mlof Ad1 and Ad2 (each at 10 mM) in separate ligation reactions at 16° C.for 18 hrs with 50 Units/ml T4 ligase. Thereafter 1 ml of 0.2M EDTA wasadded, the mixture heated at 70° C. for 5 min to inactivate the ligase,and the product stored at −70° C.

Subtractive Hybridization and PCR Amplification:

600 ng driver (iv) ds cDNA was added to each of two tubes containing 20ng Ad1-and Ad2-ligated pv cDNA. The samples were mixed, precipitatedwith ethanol, resuspended in hybridization buffer, overlaid with mineraloil and denatured/annealed in standard fashion. The two independentsamples were then combined, 200 ng fresh driver cDNA added to allow forfurther enrichment of differentially expressed mRNAs, and the mixtureagain denatured and annealed for 10 hrs at 68° C. The final sample wasdiluted in Hepes buffer with EDTA and stored at −20° C.

After subtraction two PCR amplifications were performed on thesubtracted cDNA. In the first 1 ml of subtracted cDNA was amplifiedusing 1 ml each of P1 and P2. The conditions for amplification were asdescribed by Diatchenko. The amplified products were diluted 10-fold indeionized water and 1 ml of product used for further amplification usingthe nested primers (NP1 and NP2) and a 10-cycle amplification reaction.Aliquots of the original driver/tester and subtracted cDNAs were usedfor PCR reactions with control oligonucleotide primers (β-actin) forknown “housekeeping genes”, and with primers for genes whose expressionhas been previously documented to be different in iv/pv immunized mice.These data are shown in FIGS. 1 and 2.

FIG. 1 shows PCR validation of suppressive subtractive hybridization.Samples from unsubtracted (lanes 1, 3, 5 and 7) or subtracted (lanes 2,4, 6 and 8) mRNA were reverse transcribed and tested in PCR with b-actinprimers for different PCR cycle times. Lanes 1 and 2: 15 cycles; lanes 3and 4: 20 cycles; lanes 5 and 6: 25 cycles; lanes 7 and 8: 30 cycles.

FIG. 2 shows PCR validation of suppressive subtractive hybridization.Samples from unsubtracted (lanes 2 and 4) or subtracted (lanes 3 and 5)mRNA were tested as in FIG. 1, except primers used were for IL-10, anddifferent cycle times are shown. Lanes 2 and 3: 20 cycles; lanes 4 and5: 30 cycles, lane 1: mol. wt. standard.

In addition, cloning of the subtracted cDNA was performed as follows.

Cloning and Further Analysis of Subtracted cDNA:

The PCR amplified cDNA was cloned with a TA cloning kit (Invitrogen,California) by directly ligating into the PCR II vector. Ligation wasperformed at an insert:vector ratio of 3:1 in 1× ligation buffer with T4ligase (3 U/ml) overnight at 14° C. Ligation products were then insertedinto INFaF′ competent Escherichia Coli using a standard transformationprotocol, and selected with ampicillin on plates containing X-gal(5-bromo-4-chloro-3-indolyl-D-galactoside). Miniprep plasmid DNA waspurified with a Plasmid extraction Spin kit (Qiagen, Germany) and cutwith EcoR I restriction enzyme to determine whether the plasmidscontained the expected insert. Plasmids with inserts were sequenced bythe dideoxy sequencing method using a T7 sequencing kit (PharmaciaBiotech, Canada). Nucleic acid homology searches were performed usingthe BLAST program at the National Center for Biotechnology Information(NIH, Bethesda, USA).

Further analyses of cloned material, using Northern hybridization, wasas follows. Inserts in pCRII were amplified for 12 cycles using nestedPCR primers. The amplified material was purified using Qiaquick Spin PCRPurification Kits (Qiagen), ³²P-labeled by random priming, and used as aprobe for Northern hybridization with 20 mg samples of the original (andfresh) iv or pv total RNA. Hybridization was performed in 5 ml ofExpressHyb solution (Clontech) with a minimum of 5×10⁶ cpm per 10 ngcDNA probe and 0.1 mg/ml sonicated heat-denatured salmon sperm DNA.Filters were washed 4 times, each at 15 min at 27° C. with 1×SSC and0.1% SDS, followed by a high stringency wash at 42° C. for 30 min with0.2×SSC and 0.1% SDS. Exposure times varied from 18 hrs to 6 days. FIG.3 shows an autoradiograph using ^(32g)P-labeled probes prepared from 4clones obtained using the subtraction hybridization approach describedabove (with pv cDNA as tester material and iv cDNA as driver). A labeledcontrol probe was prepared with a PCR amplicon for mouse β-actin. TotalRNA was prepared from mice receiving iv or pv immunization andequivalent amounts loaded in replicate lanes as shown, with gelsdeveloped from 18 hours (clone #28) to 6 days (#71). Clone 8 is mosthomologous with mouse poly (A) binding protein. Clone 16 is mosthomologous with rat MRC OX-2. Clone 28 is most homologous with humanzinc-finger protein. Clone 71 has no homologous sequence.

Western Blotting Protocol:

The technique used was essentially that described by Sandhu et al.(1991) as modified by Bronstein et al. (1992). Samples were obtained 14days post renal transplantation, using groups described in FIG. 5. Freshrat thymus cells were used as control. Samples were electrophoresed in12% SDS-PAGE and transferred to PVDF membranes (Novex Co., San Diego,Calif.) prior to addition of primary antibody. A commercial anti-ratOX-2 was used as test reagent; control antibody was an antibody to mouseCD8a. The developing antibody used was a commercial horse-radishperoxidase labeled anti-mouse IgG. All reagents were obtained fromCedarlane Labs (Hornby, Ontario, Canada).

DNA Sequence Homology Comparison:

Comparison of mouse OX-2 with known cDNA sequences for B7-1, B7-2, CD28and CTLA-4 was performed using a DNASIS program (version 2.0).

Results

Evaluation of Suppression Subtraction Hybridization (SSH) Technique

In order to evaluate the efficacy of the SSH technique used, theinventor used his previous evidence that, by PCR analysis, increasedexpression of mRNA for IL-10 genes was evident in lymphoid tissue frompv immunized mice. Accordingly, a dilution analysis of cDNA from thetester, driver and subtracted material, using PCR primers for β-actinand IL-10 was performed. As shown in FIG. 1, after SSH there was adetectable signal for b-actin in subtracted material only after 35cycles of amplification. By contrast, a signal was present in theunsubtracted material after only 15 cycles. Using additionalquantitative measures of template, it was found to correspond to some1000-10,000 depletion of β-actin mRNA. In a separate study, analyzingIL-10 mRNA (FIG. 2), significant enrichment of IL-10 mRNA was found asdetermined by comparison of the amplification detected at 30 cycles insubtracted/unsubtracted material (see lanes 4 and 5, FIG. 2).

In a further test of the efficiency of subtraction the mixture ofunsubtracted and subtracted tester (pv) cDNA was labeled and hybridizedto Northern blots of iv (tester) and pv (driver) total RNA. The results(data not shown) indicated that the subtracted tester cDNA probe didindeed produce a significantly stronger signal with the tester RNA.Given the evidence that for any cDNA species to produce a signal in aNorthern blot it must represent a concentration greater than 0.1-0.3% ofthe cDNA mixture, these data are again consistent with our havingproduced a high level of enrichment of pv-specific cDNA, with aconcomitant reduction in abundant cDNAs common between tester (pv) anddriver (iv) material.

Detection of Unique cDNA Fragments in Tissue from pv Immunized Mice

The efficiency and validity of SSH for detection of cDNAs unique to thetissue sample from the pv immunized mice was further confirmed aftercloning and sequence analysis of selected tester-specific cDNAs. 10randomly selected cDNA clones (of 66 sequenced) were used to probemultiple preparations of pv or iv whole RNA. All revealed unique mRNAsexpressed preferentially in the pv samples. Autoradiograms from 4 ofthese Northern blots, along with a b-actin probe as control, are shownin FIG. 3. Exposure times from 18 hrs to 6 days were used which wereinterpreted as indicative of pv specific cDNAs of different abundance inthe samples of interest.

The cDNA inserts of the 4 clones shown, along with the other 62 clones,were partially sequenced and analyzed for homology in the GenBank andEMBL data bases. A summary of these data are shown in Table 1. Note thatsome 30 cDNA fragments had at least 50% homology (BLAST score >250 overat least 50 nt) with other described sequences. A further 14 clonesshowed similar homology with known rat/human genes. Both sets mayrepresent members of different gene families. An additional 22 clonesdemonstrated no significant matches with entries in the database, andthus may represent novel genes up-regulated after pv immunization. Thatthe data shown are a minimal estimate of such differentially expressedgenes is evident from the fact that homology with IL-4 or IL-10 genesequences (mRNAs known to be over-expressed following pvimmunization—see also FIG. 2) were NOT detected in any of the 66 clonesanalyzed.

The sequence homology for the clones shown in FIG. 3 (>80% homology overthe compared sequence) led to the further characterization of theseclones. Clone 8 was shown to be most homologous with mouse poly (A)binding protein; clone 16 was shown to be most homologous with rat MRCOX-2; and clone 28 was shown to be most homolgous with human zinc-fingerprotein. No homologous sequence was found for clone 71. In the data thatfollows, the analysis of one of these clones which showed homology to arat cDNA (for OX-2, a molecule previously characterized as beingpreferentially expressed on rat thymocytes and dendritic cells) isdescribed. The rationale for further investigation of this clone lies indata showing that infusion of dendritic cells via the portal vein is apotent method for prolonging allograft survival in our model systems.Note, however, that while the bone marrow derived dendritic cells thatwere infused via the portal vein themselves express OX-2 (see above),identical data has been obtained in Northern gels to those shown in FIG.3 using tissue harvested from mice receiving, as the earliest studies(1-5) irradiated spleen cells (OX-2⁻ by FACS analysis) via the portalvein. In addition, in both situations, OX-2 mRNA was not detected bythis suppression subtraction hybridization approach when we used tissueharvested at 0.5-2.5 days post transplantation. These results areconsistent with the idea that the OX-2 signal detected is a result ofnovel increased expression in cells following pv immunization.

Probing a cDNA Library from Tissue from pv Immunized Mice for Expressionof the Murine Equivalent of Rat OX-2

A cDNA library was constructed from mRNA prepared from a pool of 5 C3Hmice receiving pv immunization with 25×10⁶ irradiated (2000 Rads)C57BL/6 bone marrow cells followed by renal transplantation as describedin the Materials and Methods, using a kit purchased from ClonTech.Clones were plated in LB medium and probed with the ^(32g)P-labeledamplicon described in FIG. 3 as showing homology with rat OX-2. A 1.3 Kbclone was detected, amplified, and shown after ³²P labeling to detect adifferentially expressed product by Northern gel analysis. Aftersequencing using an automated DNA sequencer and fluorescent-labeleddeoxynucleotides, this 1.3 Kb fragment was found to share >95% homologywith the region encoding the 3′untranslated region of the rat OX-2 mRNAas determined from the GeneBank sequence for rat OX-2.

Using a primer construct program, a 5′PCR primer representing positions1-19 of the rat GeneBank sequence (corresponding to a portion of the5′untranslated region, and the leader sequence) and 3′ primers from ourcharacterized mouse sequence were synthesized, and long-distanceamplification performed to produce an amplicon predicted to encode theopen-reading-frame (ORF) of the murine equivalent of the rat OX-2 gene.This amplicon was determined (as expected) to be of some 1.4 Kb length.Automated sequencing produced a full-length sequence for the mousehomologue of the rat MRC OX-2 gene, including an ORF with >90% homology(predicted amino acid sequence) with the corresponding rat product,along with the 3′untranslated region. This sequence has been submittedto the Genebank (accession number AF004023).

Using a DNASIS program the predicted mouse protein sequence has some 51%homology with B7-1 and B7-2, 48% with CD28 and 54% with CTLA4(unpublished).

Evidence for an Important Role for the Expressed OX-2 Homologue inProlonged Graft Survival Following pv Immunization

In an attempt to define the potential importance of the product encodedby the OX-2 gene we used a commercial antibody to rat OX-2 in atransplant model in mice receiving pv immunization and renaltransplantation. In the first such study, it was asked whether there wasevidence for specifically increased expression of the OX-2 moleculefollowing pv immunization. By FACS analysis, using dual staining ofhepatic mononuclear cells and spleen cells with OX-2 and NLDC145,similar numbers of NLDC145⁺ cells in liver or spleen samples from iv andpv immunized mice were found, (5×10⁵ and 6.5×10⁶ respectively), but a4-fold increase in the numbers of OX-2+NLDC145⁺ following pvimmunization. FIG. 4 shows a flow cytometry profile of spleen adherentcells from iv immunized/grafted mice (panels A and B) or pvimmunized/grafted mice (panels C and D). Cells were harvested 7 daysafter transplantation and stained with NLDC145 and F(ab′)₂FITC-anti-ratIgG, as well as with control (clone 107.3) mouse IgG1 serum (left handpanels) or anti-OX-2 (right hand panels) and F(ab′)₂ PE-anti-mouse IgG.Data are representative of one of three different studies. Values shownrepresent the total cell population in each quadrant. The absolutenumbers (×10⁵) of double positive cells in the liver or spleen of pvimmunized mice were 3.2±0.5 and 39±8 respectively (see FIG. 4 for FACSprofiles of spleen adherent cells). This 4-fold increase was seenregardless of the cells used for pv immunization, either bone marrowderived dendritic cells (some 20% OX-2⁺—see above) or irradiated wholespleen lymphoid cells (OX-2⁻), suggesting that they were not merelydetecting surviving OX-2⁺ (donor) cells, but novel expression of OX-2 invivo.

Western blot, FIG. 5, shows increased expression of OX-2 antigen afterpv immunization. The technique used for Western blotting is previouslydescribed. Samples were obtained 14 days post renal transplantation,using the groups described in FIG. 6. Fresh rat thymus cells (lane 5)were used as control. Lanes 1 and 2 represent samples pooled from 3donors/group (iv immunized; pv immunized+infusion of anti-OX-2respectively). Samples in lanes 3 and 4 are from individual micereceiving pv immunization and renal transplantation only (no antibodytreatment). Staining with anti-rat MRC OX-2 is shown in FIG. 5B; with acontrol antibody (to mouse Ly2.1), anti-mouse cD8a, shown in FIG. 5A.The developing antibody used was a commercial horse-radish peroxidaselabeled anti-mouse IgG. No signal was seen using the mouse IgG1 isotypecontrol clone 107.3 (BALB/c anti-TNP)-data not shown. Data arerepresentative of 1 of 3 equivalent studies.

Western blotting (see FIGS. 5A and 5B) of samples prepared from thespleen of iv vs pv immunized and grafted mice 14 days following renaltransplantation revealed staining of a band migrating with estimatedmolecular weight 43 Kd, in agreement with data elsewhere reportingextensive glycosylation of this molecule in isolates from rat thymus. Inmice receiving pv immunization along with in vivo treatment withanti-OX-2, no detectable signal was seen in Western blots (see lane 2,FIG. 5). No staining was seen with a murine IgG1 isotype control (BALB/canti-TNP, clone 107.3: unpublished), making it unlikely that the bandobserved was Fc receptor.

FIG. 6 is a graph showing percent survival versus days post renaltransplantation. Commercial anti-OX-2 monoclonal antibody, but notanti-mouse CD28 or anti-mouse CTLA4, reverses the graft prolongationfollowing donor-specific pv immunization. Groups of 6 C3H mice receivedC57BL/6 renal allografts with no other treatment (cyclosporin A only, oradditional pv immunization with 15×10⁶ C57BL/6 bone marrow deriveddendritic cells as described previously. Subsets of these latter micereceived iv injection (every second day×4 injections) with 100 mg/mouseof a commercial anti-rat OX-2 monoclonal antibody or the isotypecontrol, or of antibodies to mouse CD28 or CTLA4. The animal survivalfor the different groups shown are pooled from 2 studies. Note that themouse isotype control itself produced no modification of the increasedrenal graft survival following pv immunization. *p<0.02, Mann-WhitneyU-test).

In two final studies mice received pv immunization and transplantationas before, but now also received iv injection with commercial anti-ratOX-2 (×4 injections; 100 mg/mouse at 2 day intervals). As shown in FIGS.5A and B and 6 these infusions of anti-OX-2 significantly decreased theprolonged graft survival (FIG. 6) and increased expression of OX-2antigen (Western blotting-FIGS. 5A and 5B) seen following pvimmunization. No perturbation of graft survival following pvimmunization was seen using additional treatments withanti-CD28/anti-CTLA4 (see FIG. 6), or, in studies not shown, usinganti-B7-1 or anti-B7-2. Again infusion of the IgG1 isotype control Mab(clone 107.3) did not alter the increased graft survival seen followingpv immunization (see FIG. 6).

In separate experiments cells were harvested from mice receiving pvimmunization along with additional treatment with monoclonal antibodiesas show (see Table 2). Following treatment with anti-OX-2 there was nolonger the altered cytokine production (with polarization to productionof IL-4 and IL-10) which the inventor has described in multiple modelsystems in which animals received pv donor-specific pre-transplantimmunization. Treatment with any of the other 4 monoclonal antibodiestested did not produce this reversal in polarization of cytokineproduction seen following pv immunization-indeed, using these Mabs alonein the absence of pv immunization produced a trend to increased graftsurvival (not shown) and significant polarization in cytokine productionto increased IL-4 and IL-10 production, akin to that produced by pvimmunization itself (upper half of Table 2).

OX-2 is a molecule previously characterized by Barclay et al. (1981,1982) as being preferentially expressed on rat thymocytes and dendriticcells. Dendritic cells are known to be important signalling cells forlymphocytes, which also potentially regulate cytokine production andgraft rejection, and infusion of dendritic cells is a potent means ofinducing pv tolerance. The inventor has determined that OX-2 expressionincreased following pv immunization, and further studied whether thishad any functional consequences. As shown in FIGS. 4 and 5, there isindeed significantly increased expression of OX-2 in spleen cellsisolated from pv immunized mice, along with the increased graft survivaland polarization in cytokine production (FIG. 6 and Table 2). Incontrast, in vivo infusion of anti-OX-2 abolishes increased expressionof this molecule, simultaneously reversing the increased graft survivaland altered cytokine profile seen. This data is consistent with thepossible function of OX-2⁺ cells in promoting allograft survival.

In the studies described the donor dendritic cells infused via theportal vein were themselves OX-2⁺ (see description of materials andmethods above). However, identical data in FACS analysis (FIG. 4) andWestern Blots (FIG. 5), and from suppression subtraction hybridization(FIG. 3), have been obtained in studies in which we used irradiatedwhole spleen cells (OX-2⁻ by FACS) for pv infusion. This is consistentwith the lack of evidence for increased mRNA expression of OX-2 early(1-2 days) post transplant, as noted above. Thus it seems most likelythat an operationally important “OX-2 signal” detected in the spleen ofthe pv immunized mice can derive from new expression, rather thannecessarily from infused OX-2⁺ cells. In the absence of a polymorphicmarker for OX-2, however, it cannot be determined whether increasedexpression is from donor or host cells (or both). Indeed, it is perhapssomewhat surprising that the murine antibody to rat OX-2 cross-reacts inthe fashion shown with murine OX-2. Definitive analysis of the in vivorole of OX-2 awaits similar studies to those above, using antibodiesdeveloped against the murine OX-2 homologue—these experiments arecurrently in progress. It is also important to point out that while pvimmunization led to only a 4-fold alteration in the absolute number ofdetectable OX-2⁺ NLDC⁺ cells in the spleen/liver (see text and FIG. 4),nevertheless in the face of this 4-fold difference a clear difference inOX-2 signals in Northern gels using RNA from pv vs iv immunized mice(FIG. 3), along with evidence for a role for this quantitativedifference in the outcome of graft survival (FIG. 6) were detected.Presumably these results reflect respectively the limitation to thesensitivity of the Northern assay used, and some function of thequantitation of “co-stimulation” occurring after OX-2:OX-2 ligandinteraction.

While there was some 50% homology of the predicted protein sequence ofmurine OX-2 with murine B7-1, B7-2, CD28 and CTLA4 (Borriello et al.,1997), antibodies to the latter molecules did not reverse the prolongedgraft survival and altered cytokine production following pv immunization(FIG. 6, Table 2—see also (Castle et al., 1993)). In fact these latterantibodies themselves, infused in the absence of pv immunization,produced some of the same changes in cytokine production induced by pvimmunization (Table 2).

Example 2 Murine OX-2

This example describes the cloning and sequencing of murine MRC OX-2.

A cDNA library was constructed from MLN cells derived from adult C3Hmice, preimmunized 5 days earlier with 10×106 allogeneic B10.BR bonemarrow-derived dendritic cells allogeneic cells by the portal venous(pv) route, using a Cap Finder PCR cDNA library construction kit(Clontech). The inventor had previously isolated, using a PCR-SelectcDNA subtraction hybridization kit (Clontech) and RNAs obtained frompooled MLN of mice immunized by the pv route or via the lateral tailvein (iv), a 350 bp amplicon which showed over 98% homology with the 3′untranslated region of rat MRC OX-2 cDNA. Northern blot analysisconfirmed that this amplicon detected a differentially expressed productin RNAs prepared from iv vs pv immunized mice. This amplicon was used toscreen 5×10⁵ clones of the amplified library. The sequences of cDNAclones were established with an Applied Biosystems 377 AutomatedSequencer, utilizing the Dye Terminator Cycle Sequencing method (AppliedBiosystems, Foster City, Calif.). The nucleotide sequence reported inthis paper has been submitted to the GenBank/EMBL Data Bank withaccession number AF004023.

The cDNA shown in FIG. 7 has an open reading frame of 837 base pairs,and a deduced amino acid sequence (FIG. 8) of 248 amino acids, of which30 represent a cleaved leader sequence. The predicted molecular weightof this, and the equivalent molecules in rat and human, is approximately25 kDa. The measured molecular weight in rat thymocytes, where themolecule is highly gylcosylated, is 47 kDa.

The murine MRC OX-2 shows some 92% and 77% homology overall at the aminoacid level with equivalent molecules in rat or human respectively. Asnoted for the rat molecule, the sequence from a 203-229 seems likely torepresent a membrane spanning domain (highly hydrophobic region), whilethe region from 229-248 is likely the intracytoplasmic region, with astretch of highly basic residues immediately C-terminal to position 229.Homology in the combined transmembrane and C-terminal regions with ratand human shows some 98% and 85% similarity respectively. As predictedfrom membership in the Ig supergene family, there are a number ofconserved Cys residues forming the disulphide bonds between b-strands ofIg-like domains, (21 and 91; 130 and 184 respectively); residue 91 waspreviously found to be the most highly conserved among members of theimmunoglobulin superfamily. Homology between the N-terminal Ig-domainwith rat and human, versus the next Ig-domain, is 88% and 82%, or 97%and 73% respectively. This relative concentration in variability betweenrat and mouse in the V-terminal Ig-domain may be more understandablewhen the ligand specificity for the molecules in these species isclarified. Note that the presumed extracellular portion of the molecule(1-202) contains a number of sites for N-glycosylation which arepreserved across species (44, 65, 73, 80, 94, 127, 130 and 151). Thiswas previously reported for the rat cDNA sequence, and inferred from themeasured size of the expressed material in rat thymocytes.

The intracytoplasmic region of the molecules has no sequence identitywith known signaling kinases, nor does it have the well-describedconsensus sequence for the immunoreceptor tyrosine activation motif(ITAM: DXXYXXLXXXXXXXYDXL). In addition, it lacks typical SH2 or SH3domains to serve as “docking sites” for adapter molecules which might inturn co-opt other protein kinases in an activation cascade. Accordinglythe ligand-binding activity of the extracellular domains presumablyrepresent the biologically important region of the molecule. Somepossible functions attributable to ligand interaction with OX-2 can beinferred from other data in the literature. A homologous molecule,Ng-CAM, has been reported to bind a protein-tyrosine phosphatase viaN-linked oligosaccharide residues, and protein tyrosine phosphatases areknown to play a key regulatory role in immune responses. More recentlyALCAM, another adhesion molecule member of the Ig superfamily, the genefor which is located close to that for OX-2 on chromosome 3 in humans,has been shown to bind CD6 (a member of the scavenger receptor cysteinrich family, SRCR), and antibodies to CD6 may themselves play a role inregulating immune function.

Example 3 OX-2 Positive Cells Inhibit Type-1 Cytokine Production

The inventor has shown that hepatic mononuclear, non-parenchymal, cells(NPC) can inhibit the immune response seen when allogeneic C57BL/6dendritic cells (DC) are incubated with C3H spleen responder cells.Cells derived from these cultures transfer increased survival of C57BL/6renal allografts in C3H mice. The inventor also found that increasedexpression of OX-2 on dendritic cells was associated with inhibition ofcytokine production and renal allograft rejection. The inventor furtherexplored whether inhibition by hepatic NPC was a function of OX-2expression by these cells.

Fresh C57BL/6 spleen derived DC were cultured with C3H spleen respondercells and other putative co-regulatory cells. The latter were derivedfrom fresh C3H or C57BL/6 liver NPC, or from C3H or C57BL/6 mice treatedfor 10 days by intravenous infusion of human Flt3 ligand (Flt3L).Different populations of murine bone-marrow derived dendritic cells fromcultures of bone marrow with (IL-4+GM-CSF) were also used as a source ofputative regulator cells. Supernatants of all stimulated cultures wereexamined for functional expression of different cytokines (IL-2, IL-4,IFNγ, TGFβ). It was found that fresh C57BL/6 splenic DC induced IL-2 notIL-4 production. Cells from the sources indicated inhibited IL-2 andIFNγ production, and promoted IL-4 and TGFβ production. Inhibition wasassociated with increased expression of OX-2 on these cells, as definedby semi-quantitative PCR and FACS analysis. By size fractionation, cellsexpressing OX-2 were a subpopulation of NLDC145+ cells. This dataimplies a role for cells expressing OX-2 in the regulation of inductionof cytokine production by conventional allostimulatory DC.

Materials and Methods

-   Mice: Male and female C3H/HEJ and B10.BR (H-2^(k/k)), B10.D2    (H-2^(d/d)) and C57BL/6 (H-2^(b/b)) mice were purchased from the    Jackson laboratories, Bar Harbour, Me. Mice were housed 5/cage and    allowed food and water ad libitum. All mice were used at 8-12 weeks    of age.-   Monoclonal antibodies: The following monoclonal antibodies (Mabs),    all obtained from Pharmingen (San Diego, Calif., USA) unless stated    otherwise, were used: anti-IL-2 (JES6-1A12; biotinylated, JES6-5H4);    anti-IL-4 (11B11, ATCC; biotinylated, BVD6-24G2); anti-IFNγ (R4-6A2,    ATCC; biotinylated XMG1.2); anti-IL-10 (JES5-2A5; biotinylated    SXC-1); PE anti-B7-1/B7-2 (Cedarlane Labs, Hornby, Ontario, Canada).

Rat anti-mouse OX-2 monoclonal antibodies were prepared byImmuno-Precise Antibodies Ltd. (Victoria, BC, Canada) followingimmunization of rats with a crude membrane extract of LPS stimulatedmurine DC, followed by fusion with a non-secreting rat myeloma parentcell line (YB2/3HI.P2.G11.16Ag.20). Hybridoma supernatants were screenedin ELISA using plates pre-coated with a 40-45 Kd preparation of DCextracts run on Western gels (Barclay, A. N. 1981. Immunology 44:727;Barclay, A. N., and H. A. Ward. 1982. Eur. J. Biochem. 129:447).Positive clones were re-screened using FACS analysis of CHO cellstransduced with a cDNA clone encoding full-length murine OX-2 (Chen, Z.,H. Zeng, and R. M. Gorczynski. 1997. BBA. Mol. Basis Dis. 1362:6-10).FITC-conjugated F(ab′)2 rabbit anti-rat IgG (non cross-reactive withmouse IgG) from Serotec, Canada was used as second antibody. The Mabselected for further analysis (M3B5) was grown in bulk in a CELLMAXsystem (Cellco Inc., Germantown, Md.). A crude preparation of ratimmunoglobulin (30% saturated ammonium sulphate preparation) was used asa control Ig.

In tissue culture assays where anti-cytokine Mabs were used to confirmthe specificity of the assay used 10 μg/ml of the relevant Mabs wasfound to neutralize up to 10 ng/ml of the cytokine tested.

NLDC145 (anti-mouse DC) was also obtained from Serotec. Recombinantmouse IL-4 was a kind gift from Dr. L. Yang (The Toronto Hospital);mouse rGM-CSF was purchased from Pharmingen. Recombinant human Flt3L(derived from CHO cells) was a kind gift from Dr. A. B. Troutt, ImmunexCorp., Seattle, Wash., USA.

Renal Transplantation

Renal transplantation was performed essentially as described elsewhere(Gorczynski, R. M. et al. 1994a. Transplantation 58:816-820). Animalswere anesthetized with a combination of halothane and nitrous oxideinhalation, using novogesic for post-op analgesia. Orthotopic renaltransplantation was performed using routine procedures. In brief, Donoranimals received 200 Units of heparin, and kidneys were flushed with 2ml of ice cold heparinized physiological saline solution, prior toremoval and transplantation into recipient animals with leftnephrectomy. The graft renal artery was anastomosed to the recipient'sabdominal aorta, and the renal artery was anastomosed to the recipient'sinferior vena cava. The ureter was sewn into the recipient bladder usinga small donor bladder patch. All recipients received im injection withcefotetan (30 mg/Kg) on the day of transplantation and for 2 succeedingdays. The remaining host kidney was removed 2 days aftertransplantation, unless otherwise indicated. Treatment of recipientswith pv immunization, by monoclonal antibodies, or by oral immunizationwas as described in individual studies.

Portal Vein and Oral Immunization

Portal vein and oral immunization was performed as described earlier(Gorczynski, R. M. 1995a. Cell. Immunol. 160:224-231; Gorczynski, R. M.et al. Transplantation 62:1592-1600). All animals were anaesthetizedwith nembutal. A midline abdominal incision was made and the visceraexposed. Cells were injected in 0.1 ml through a superior mesentericvein using a 30 gauge needle. After injection the needle was rapidlywithdrawn and hemostasis secured without hematoma formation by gentlepressure using a 2 mm 3 gel-foam.

Bone-marrow derived dendritic cells (DC) for pv immunization wereobtained by culture of T depleted bone marrow cells in vitro with rIL-4and rGM-CSF (Gorczynski, R. M. et al. Transplantation 62:1592-1600).Staining with NLDC145 and FITC anti-rat IgG, or with FITC anti-CD3confirmed >95% NLDC145+ and <5% CD3+ cells at day 10 of culture(Gorczynski, R. M. et al. Transplantation 62:1592-1600). These cellswere washed and injected into mice or used for mixed leucocyte cultures.

Preparation of Cells:

Spleen and bone marrow (Gorczynski, R. M. et al. Transplantation62:1592-1600) cell suspensions were prepared aseptically from individualmice in each experiment. Hepatic mononuclear nonparenchymal cells (NPC)were isolated essentially as described elsewhere (Gorczynski, R. M.1994b. Immunology 81:27-35). Tissue was first digested at 37° C. for 45min with a mixture of collagenase/dispase, prior to separation (15 minat 17,000 rpm at room temperature) over mouse lymphopaque (CedarlaneLabs). Mononuclear cells were resuspended in a-Minimal Essential Mediumsupplemented with 2-mercaptoethanol and 10% fetal calf serum (αF10).Where cells were obtained from Flt3L injected mice, animals were treatedby iv injection of 10 mg/mouse Flt3L daily for 10 days. After enzymedigestion recovery of liver/spleen cells from these mice was markedlyincreased compared with saline-injected controls (120×10⁶, 390×10⁶ vs7×10⁶ and 120×10⁶ respectively).

Cytotoxicity and Cytokine Assays:

In cultures used to assess induction of cytotoxicity or cytokineproduction responder cells were stimulated with irradiated (2000R)stimulator cells in triplicate in αF10. Supernatants were pooled fromreplicate wells at 40 hrs for cytokine assays (below). No reproducibledifferences in cytokine levels have been detected from cultures assayedbetween 36 and 54 hrs of stimulation. In some experiments the culturesreceived 1 mCi/well (at 72 hrs) of ³HTdR and proliferation was assessedby harvesting cells 14 hrs later and counting in a well-type b-counter.

Where cytoxicity was measured cells were harvested and pooled fromequivalent cultures at 5 days, counted, and recultured at differenteffector:target with ⁵¹Cr EL4 (H2^(b/b)) or P815 (H2^(d/d)) tumor targetcells. Supernatants were sampled at 4 hrs for assessment of specificcytotoxicity.

IL-2 and IL-4 activity were assayed by bioassay using the IL-2/IL-4dependent cell lines, CTLL-2 and CT4.S respectively. Recombinantcytokines for standardization of assays was purchased from Genzyme(Cambridge, Mass.). IL-2 assays were set up in the presence of 11B11 toblock potential stimulation of CTLL-2 with IL-4; IL-4 assays were set upin the presence of S4B6 to block IL-2 mediated stimulation. Both theIL-2 and IL-4 assays reproducibly detected 50 pg of recombinantlymphokine added to cultures.

In addition, IL-2, IL-4, IFNγ and IL-10 were assayed using ELISA assays.For IFNγ the assay used flat-bottomed Nunc plates (Gibco, BRL) coatedwith 100 ng/ml R4-6A2. Varying dilutions of supernatant were bound intriplicate at 4° C., washed ×3, and biotinylated anti-IFNγ (XMG1.2)added. After washing, plates were incubated with streptavidin-horseradish peroxidase (Cedarlane Labs, Hornby, Ontario), developed withappropriate substrate, and OD₄₀₅ determined using an ELISA plate reader.Recombinant IFNγ for standardization was from Pharmingen. IL-10 wassimilarly assayed by ELISA, using JES5-2A5 as a capture antibody andbiotinylated SXC-1 as developing antibody. rIL-10 for standardizationwas from Pepro Tech Inc. (Rocky Hill, N.J.). Each assay detected 0.1ng/ml cytokine. ELISA assays for IL-2 and IL-4 used JES6-1A12 and 11B11as capture antibodies, with JAS6-5H4 or BVD6-24G2 as developingantibodies. Sensitivity of detection was 20 pg/ml for each cytokine.Where checked the correlation between bioassay and ELISA for IL-2 orIL-4 was excellent (r>0.90). In all studies reported below, data areshown from ELISA assays only. Where cytokine data are pooled fromseveral studies (e.g. FIGS. 14, 16, 17), absolute values of cytokineproduction were obtained as above using commercial recombinant cytokinesto standardize the assays. In our hands, supernatants fromC3Hanti-C57BL/6 cultures, under the conditions described, reproduciblycontain 950±200 and 80±25 pg/ml IL-2 and IL-4 respectively.

Preparation of RNA:

Different sources of tissue from renal-grafted female mice receiving DCand kidney allografts from male mice were harvested for RNA extractionas described elsewhere (Gorczynski, R. M. 1995a. Cell. Immunol.160:224-231). The OD280/260 of each sample was measured and reversetranscription performed using oligo (dT) primers (27-7858: Pharmacia,USA). The cDNA was diluted to a total volume of 100 ml with water andfrozen at −70° C. until use in PCR reactions with primers for murineGAPDH, B7-1, B7-2 or OX-2. The sense (S) and antisense (AS) primers weresynthesized by the Biotechnology Service Centre, Hospital for SickChildren, Toronto, using published sequences. 5′ primers were ³²Pend-labeled for PCR and had comparable levels of specific activity afterpurification by ethanol precipitation. 5 ml cDNA was amplified for 35cycles by PCR, and samples were analyzed in 12.5% polyacrylamide gelsfollowed by overnight (18 hrs) exposure for autoradiography. In controlstudies, using H-Y primer sets, this technique reliably detects H-Y mRNAfrom extracts of female spleen cells to which male cells are added at aconcentration of 1:105 (Gorczynski, R. M. 1995a. Cell. Immunol.160:224-231; Gorczynski, R. M. et al. Transplantation 62:1592-1600).Quantitative comparison of expression of different PCR products useddensitometric scanning of the autoradiograms.

GAPDH Sense: (SEQ ID NO: 10) 5′TGATGACATCAAGAAGGTGGTGAAG3′GAPDH Antisense: (SEQ ID NO: 11) 5′TCCTTGGAGGCCATGTAGGCCAT3′ B7-1 Sense:(SEQ ID NO: 12) 5′CCTTGCCGTTACAACTCTCC3′ B7-1 Antisense: (SEQ ID NO: 13)5′CGGAAGCAAAGCAGGTAATC3′ B7-2 Sense: (SEQ ID NO: 14)5′TCTCAGATGCTGTTTCCGTG3′ B7-2 Antisense: (SEQ ID NO: 15)5′GGTTCACTGAAGTTGGCGAT3′ OX-2 Sense: (SEQ ID NO: 16)5′GTGGAAGTGGTGACCCAGGA3′ OX-2 Antisense: (SEQ ID NO: 17)5′ATAGAGAGTAAGGCAAGCTG3′Statistical Analysis:

In studies with multiple groups, ANOVA was performed to comparesignificance. In some cases (as defined in individual circumstances)pairwise comparison between groups was also subsequently performed.

Results

Antigen Stimulation, in the Presence of Hepatic NPC, Induces Developmentof a Cell Population Capable of Inhibiting Proliferation and IL-2Production on Adoptive Transfer:

In a previous manuscript (Gorczynski, R. M. et al., Transplantation. 66:339-349) it was reported that C3H spleen cells stimulated in thepresence of syngeneic NPC and allogeneic (C57BL/6) DC produced a cellpopulation able to inhibit generation of IL-2 from fresh spleen cellsstimulated with C57BL/6 DC, and capable of inhibiting C57BL/6 renalallograft rejection in vivo. In order to ask whether this function ofNPC was MHC restricted or not, the following study was performed.

C57BL/6 (H2^(b/b)) spleen cells were stimulated in vitro with B10.BR(H2^(k/k)) bone-marrow derived DC, in the presence/absence of thefollowing NPC: C57BL/6; B10.BR; B10.D2 (H2^(d/d)). In addition, controlcultures were incubated with the NPC only. Proliferation and IL-2/IL-4production was measured in one aliquot of these primary cultures. Inaddition, at 5 days, cells were harvested from another set of theprimary cultures, washed, and 2×10⁵ cells added to cultures containing5×10⁶ fresh C57BL/6 spleen cells and B10.BR DC. Proliferation andcytokine production was measured in these latter cultures in standardfashion. Data pooled from three equivalent studies are shown in panelsA) and B) of FIG. 9.

FIG. 9 is a bar graph showing regulation of proliferation and cytokineproduction following stimulation by allogeneic DC using hepatic NPC inaccordance with the methods described herein. In panel A) cultures wereinitiated with 5×10⁶ C57BL/6 responder spleen cells alone (group 1), orwith 2×10⁵ B10.BR DC (group 2). Further groups (3-5, and 6-8respectively) contained C57BL/6 responder cells and 2×105 NPC fromeither C57BL/6, B10.D2 or B10.BR respectively (3-5) or these same NPCand B10.BR DC (6-8). Data show mean proliferation and cytokineproduction from triplicate cultures in three separate studies. In panelB) data show proliferation and cytokine production from cultures of5×10⁶ C57BL/6 responder spleen cells stimulated in triplicate with 2×105B10.BR DC alone, or with the addition also of 2×10⁵ cells harvested fromthe cultures shown in the upper panel. Again data represent arithmeticmeans of 3 separate experiments. *p<0.05 compared with control cultures(far left in each panel).

There are a number of points of interest. As previously documented,addition of NPC syngeneic with spleen responder cells (C57BL/6 in thiscase) to cells stimulated with allogeneic (B10.BR) DC led to decreasedproliferation and IL-2 production from those responder cells comparedwith cells stimulated by DC alone (compare groups 6 and 2 of upper panelof FIG. 9, panel A). IL-4 production in contrast was enhanced. NPCalone, whether syngeneic or allogeneic to the responder cells, producedno obvious effect (groups 3-5, panel A) of FIG. 9). Furthermore, cellsfrom primary cultures receiving the DC+NPC mixture were able to inhibitproliferation and IL-2 production (while promoting IL-4 production) fromfresh spleen cells stimulated in secondary cultures with the same(B10.BR) DC (see panel B) of FIG. 9). However, data in this Figure makeanother important point. The same inhibition of proliferation/IL-2production in primary cultures was seen using either B10.BR NPC (MHCmatched with the DC stimulus-group 8, panel A) of FIG. 9) or withthird-party B10.D2 NPC (MHC-mismatched with both spleen responder cellsand allogeneic stimulator DC-group 7, panel A) of FIG. 9). Again noobvious effect was seen in cultures stimulated with B10.BR or B10.D2 NPCalone (groups 4 and 5). Finally, cells taken from primary culturesstimulated with DC and NPC from either B10.BR or B10.D2 could alsoinhibit proliferation/IL-2 production from secondary C57BL/6 spleen cellcultures stimulated with B10.BR DC-again cells taken from primarycultures with NPC alone produced no such inhibition (see panel B) ofFIG. 9). Thus the inhibition of proliferation/IL-2 production andenhancement of IL-4 production seen in primary cultures, as well as theinduction of suppression measured in secondary cultures, all induced byNPC, are not MHC-restricted.

Specificity of Inhibition/Suppression Induced by Hepatic NPC:

One interpretation of the data shown in FIG. 9 and elsewhere is that NPCdeliver a signal to DC-stimulated cells which is distinct from theantigen-signal provided by the DC themselves (and is MHCnon-restricted). This signal modulates the antigen-specific signalprovided by the DC. In order to assess the antigen-specificity of theimmunoregulation described in FIG. 9, the following experiment wasperformed.

C57BL/6 spleen responder cells were stimulated with B10.D2 or B10.BRbone marrow-derived DC, in the presence/absence of NPC from B10.BR orB10.D2 mice. Proliferation and cytokine production was measured inaliquots of these cultures as before. In addition, further aliquots ofcells harvested from these primary cultures were added to cultures offresh C57BL/6 spleen cells stimulated with B10.BR (panel B)—FIG. 10) orB10.D2 (panel C)—FIG. 10) DC. Again proliferation and cytokineproduction was measured. Data pooled from three such studies are shownin FIG. 10.

FIG. 10 shows specificity of inhibition of proliferation of cytokineproduction by hepatic NPC (see FIG. 9 and description of FIG. 9 for moredetails). In panel A), 5×10⁶ C57BL/6 spleen cells were stimulated intriplicate for 3 days with 2×10⁵ B10.BR or B10.D2 DC, with/without 2×10⁵NPC derived from B10.D2 or B10.BR mice. Data shown are arithmetic meansof 3 repeat studies. In panels B) and C), fresh C57BL/6 responder spleencells were cultured in triplicate with either B10.BR DC (panel B), orB10.D2 DC (Panel C), with/without 2×10⁵ additional cells from theprimary cultures (groups 1-6 in panel A). Again data representarithmetic means of proliferation/cytokine production from 3 studies.*p<0.05 compared with control cultures (far left in each panel).

Data from the primary cultures (panel A)) recapitulates the observationsmade in FIG. 9, and show that NPC inhibit proliferation and IL-2production from DC-stimulated responder cells in an antigen andMHC-unrestricted fashion. However, the data in panels B) and C) of thisfigure show clearly that adoptive transfer of inhibition using cellsfrom these primary cultures occurs in an antigen-restricted fashion,dictated by the antigen-specificity of the DC used in the primarycultures, not of the NPC used for induction of suppression. Theseauxiliary cells in the NPC population thus have a functional property ofbeing “facilitator cells for induction of suppression”. Note that inother studies (data not shown) where the final assay system involvedmeasuring cytotoxicity to allogeneic target cells, a similar inhibitionof lysis (rather than cytokine production) was seen using cellsharvested from primary cultures stimulated with DC and hepatic NPC (seeGorczynski, R. M., et al. 1998a. Transplantation. 66: 339-349).

Hepatic Cell Preparations from Flt3L Treated Mice are a Potent Source ofDC and “Facilitator” Cells:

It has been reported at length that pv infusion of alloantigen, or ivinfusion of liver-derived allogeneic mononuclear cells inducesoperational unresponsiveness in recipient animals (Gorczynski, R. M.1995a. Cell. Immunol. 160:224-231; Gorczynski, R. M. et al.Transplantation 62:1592-1600; Gorczynski, R. M. et al. 1994a.Transplantation 58:816-820; Gorczynski, R. M., and D. Wojcik. 1992.Immunol. Lett. 34:177-182; Gorczynski, R. M. et al. 1995b.Transplantation. 60:1337-1341). The total hepatic mononuclear cell yieldfrom normal mice is of the order of 5×10⁶ cells/mouse. In order toincrease the yield, and explore the possibility that the liver itselfmight be a source both of allostimulatory DC and “facilitator” cells 2C57BL/6 mice were exposed for 10 days to daily iv infusions of 10mg/mouse human CHO-derived Flt3L, a known growth factor for DC (Steptoe,R. J. et al. 1997. J Immunol. 159:5483-5491). Liver tissue was harvestedand pooled from these donors and mononuclear cells prepared as describedin the Materials and Methods section above (mean 130×10⁶ cells/donor).These cells were further subjected to sub-fractionation by size usingunit gravity sedimentation techniques (Miller, R. G., and R. A.Phillips. 1969. J. Cell. Comp. Physiol. 73:191-198). A typical sizeprofile for recovered cells is shown in FIG. 11 (one of 3 studies).

FIG. 11 shows OX-2 expression in a subpopulation of NPC. It is asedimentation analysis (cell profile) and FACS analysis of cellsisolated at 10 days from Flt3L-treated C57BL/6 mice. Two C57BL/67 micereceived 10 mg/mouse Flt3L iv daily for 10 days. Hepatic NPC weresedimented for 3 hrs at 4° C., and the fractions shown collected (Fxs1-4 with sedimentation velocities 2.5-3.8, 3.8-5.1, 5.1-6.4 and 6.4-8.0mm/hr respectively). Aliquots of the cells were stained in triplicatewith the Mabs shown. The remainder of the cells were used as in FIGS.12-14. Data are pooled from 3 studies.

In these same studies cells isolated from the various fractions shown inFIG. 11 were tested as follows. Firstly, cells were stained withFITC-labeled Mabs to B7-1, B7-2, NLDC145 and rat anti-mouse OX-2 (M3B5)with FITC anti-rat IgG as second antibody. In addition, mRNA extractedfrom the different cell samples were assayed by PCR for expression ofGAPDH, B7-1, B7-2 and OX-2. Data are shown in FIG. 11 (pooled from 3separate studies) and FIG. 12 (representative PCR data from oneexperiment).

FIG. 12 shows PCR detection of B7-1, B7-2 and OX-2 in hepatic NPMC. Itis a PCR analysis for mRNA expression of OX-2, B7-1 and B7-2 in varioushepatic NPC cell fractions isolated from Flt3L treated mice (see FIG.11). Data are representative from 1 of 3 studies.

Further aliquots of the cells were used to stimulate fresh C3H spleenresponder cells in culture. Proliferation and cytokine assays wereperformed as before (see FIG. 9), and in addition cells were taken fromthese primary cultures and added to fresh secondary cultures of C3Hspleen responder cells and C57BL/6 bone marrow-derived DC. Againproliferation and cytokine production was assayed from these secondarycultures. Data pooled from 3 studies of this type are shown in FIG. 13(panels A) and B).

FIG. 13 shows that hepatic NPMC from Flt3L treated mice results IL-2 andIL-4 production. Stimulation of proliferation/cytokine production by NPCfrom Flt3L treated mice, and inhibition of the same (where stimulationis induced by a separate population of DC) is a function of differentcell populations. (See text and FIGS. 11-12 for more details.) HepaticNPC fractions were derived from Flt3L treated C57BL/6 mice and were usedto stimulate C3H spleen cells in triplicate cultures, alone or in thepresence of bone-marrow derived C57BL/6 DC (see panel A). Data showarithmetic means for proliferation/cytokine production from 3experiments. In addition, cells harvested from these primary cultureswere added to fresh C3H spleen cells stimulated with C57BL/6 DC (panelB), and again proliferation/cytokine production assayed. *p<0.05compared with control groups (far left of panel).

Finally, cells from the various fractions were infused iv into 2/groupC3H mice which also received C57BL/6 renal allografts as antigenchallenge. Spleen cells were harvested from these individual mice 10days after transplantation and restimulated in culture with C57BL/6 orB10.D2 DC, again with cytokines measured at 40 hrs (see FIG. 14).

FIG. 14 is a bar graph of cytokines produced from cells from C3H micewith C57BL/b renal allografts and NPC from Flt3 treated C57BL/6 donors.OX-2⁺ NPC infused iv into renal transplant allograft recipients leads topolarization of cytokine production (to IL-4, IL-10 and TGFβ) in spleencells harvested from those mice and restimulated in vitro. Fractions ofNPC from Flt3L treated C57BL/6 mice (from FIG. 11) were infused iv into2/group C3H recipients, receiving C57BL/6 renal allografts (along withCsA) in standard fashion (see Materials and Methods). Mice weresacrificed 14 days after transplantation and spleen cells stimulated invitro in triplicate with C57BL/6 DC stimulator cells. Cytokines wereassayed in the supernatants of these cultures at 60 hrs. Data showarithmetic means pooled from cultures in 3 studies of this type. *p<0.05compared with control groups (far left-no NPC infused).

Data in FIG. 11 show that distinct subpopulations of slow-sedimentingcells express OX-2 in the cells harvested from Flt3L treated mice, whencompared with cells expressing B7-1 and/or B7-2. In general expressionof OX-2 and B7-2 occurred in equivalent subpopulations.Faster-sedimenting cells (Fx 3 and 4 in FIG. 11), while staining forNLDC145, were positive by fluorescence mainly for B7-1, not B7-2 orOX-2. Similar conclusions were reached both by FACS analysis of cellpopulations (FIG. 11), and by PCR analysis of mRNA (FIG. 12).

When the functional capacity of these different cell populations wasinvestigated (FIGS. 13 and 14) it was found that optimal directstimulation (or proliferation and IL-2 production) was seen from B7-1expressing cells (Fxs 3 and 4 in panel A) of FIG. 13), while only OX-2expressing cells (Fxs 1 and 2 in FIGS. 11 and 12) were capable ofproducing the inhibitory effects defined earlier (FIGS. 9 & 10) in thetwo-stage culture system (panel B) in FIG. 13). These same cells (Fxs 1and 2) were in turn able, after iv infusion, to polarize cells from micegiven renal allografts to produce predominantly IL-4, IL-10 and TGFβproduction on restimulation in vitro (FIG. 14). These data areconsistent with the notion that after FltL treatment of mice expansionof a population of immunostimulatory DC occurs within the liver, whichalso contains another distinct population of (facilitator) cells whichpromote immunoregulation.

Evidence that Cell Populations with “Facilitator” Activity from theLiver of Flt3L Treated Mice Prolong Graft Survival In Vivo:

Since it has been reported elsewhere that there is a good correlationbetween treatments (such as pv immunization) which decrease IL-2production and increase IL-4 production from restimulated cells andprolongation of graft survival (Gorczynski, R. M., and D. Wojcik. 1994.J. Immunol. 152:2011-2019; Gorczynski, R. M. 1995a. Cell. Immunol.160:224-231; Gorczynski, R. M. et al. Transplantation 62:1592-1600), andthat increased expression of OX-2 is also independently associated withincreased graft survival after pv immunization (Gorczynski, R. M. et al.1998b. Transplantation. 65:1106-1114), the next question was whethercells isolated from Flt3L treated mice which induced inhibitory functionin vitro (see FIGS. 9, 10 and 13), and expressed increased amounts ofOX-2 (FIGS. 11, 12) were themselves capable of promoting increased graftsurvival in vivo.

Groups of 2 C57BL/6 mice received iv infusions of 10 mg/mouse Flt3L for10 days as before. Cells were isolated from the liver by enzymedigestion, and fractionated by unit gravity sedimentation. 4 pools ofcells were recovered, and an aliquot stained as before in FACS withanti-OX-2. Groups of 2 C3H mice received 10×10⁶ cells iv from the 4separate pools. A control group received saline injections only. Overthe next 48 hrs all mice received C57BL/6 renal transplants. All micereceived CsA (10 mg/Kg) on the day of renal transplantation. Data inFIG. 15 are pooled from 3 studies of this type (representing 6mice/group), and show the animal survival in these 5 different groups.

FIG. 15 shows NPC from Flt3L treated C57BL/6 mice, infused iv intorecipient C3H mice, inhibit C57BL/6 renal allograft rejection. Two micegroups received the different subpopulations of NPC derived from Flt3Ltreated mice shown in FIGS. 11 and 12. Fxs 1 and 2 were OX-2+. Micereceived C57BL/6 renal allografts within 48 hrs along with CsA (seeMaterials and Methods). Animal survival was followed as an end point.Data shown are pooled from 3 studies (6 mice/group). *p<0.05 comparedwith mice receiving CsA only.

It is quite clear from this Figure that only hepatic cells expressingOX-2 (Fxs 1 and 2—see FIGS. 11 and 12) were capable of promotingincreased graft survival after iv infusion. Comparison of these datawith those in FIG. 13 confirm that these cell populations were alsothose identified, using a 2-stage culture assay system, as cells withfunctional “facilitator” activity (see also FIGS. 9 and 10). There wasno significant difference in survival between groups receiving NPC-Fx1or NPC-Fx2 in this experiment, in keeping with relatively equivalentlevels of OX-2 expression in these fractions (FIG. 11).

Anti-OX-2 Monoclonal Antibody In Vitro Reverses Regulation Induced byHepatic NPC:

A final study was directed to whether anti-OX-2 monoclonal antibodyM3B5, added to cultures of C3H spleen responder cells, allogeneic(C57BL/6) DC and NPC from C57BL/6 mice, could prevent the inhibition ofIL-2 production in primary cultures, and the development of cells ableto inhibit such cytokine responses from freshly stimulated respondercells in secondary cultures (see FIGS. 9, 10 and 13). Data in FIGS. 16and 17 are pooled from 3 studies of this type.

FIG. 16 is a bar graph showing the effect of anti B7-1; B7-2; or OX-2 onprimary allostimulation. It shows that anti-OX-2 Mab increases IL-2cytokine production in vitro after stimulation of C3H responder spleencells with C57BL/6 DC. Subgroups of cultures contained the Mabs shown.Cytokines were assayed at 60 hrs. All data represent arithmetic meanspooled from 3 repeat studies. *p<0.05 compared with control group (farleft).

FIG. 17 is a bar graph showing that anti-OX-2 reverses inhibition byNPC. It shows that anti-OX-2 Mab inhibits development ofimmunoregulatory cells in vitro following incubation with hepatic NPC.C3H responder spleen cells were incubated in triplicate with C57BL/6 DCalong with NPC (see FIGS. 9 and 10). Subgroups of these culturescontained the Mabs shown. Cytokines were assayed in cultures at 60 hrs(panel A). In addition, cells were harvested from all groups, washed andadded to fresh C3H responder spleen cells and C57BL/6 DC (panel B).Cytokines in these groups were assayed 60 hrs later. All data representarithmetic means pooled from 3 repeat studies. *p<0.05 compared withcontrol group from cultures of NPC with no monoclonal antibodies (farleft in Figure)—see also FIG. 16.

Primary cultures were of two types, containing C3H responder spleencells and C57BL/6 DC alone (FIG. 16), or the same mixture with addedC57BL/6 NPC (FIG. 17). Subsets of these cultures contained in additioneither 5 mg/ml of anti-B7-1, anti-B7-2 or anti-OX-2. Supernatants fromresponder cells stimulated in the presence of DC only were assayed after60 hrs for cytokine production (FIG. 16). For the primary culturesincubated with both DC and NPC, supernatants were harvested at 60 hrsand tested for cytokine production (FIG. 17A). In addition, cells wereharvested after 5 days, washed, and added to secondary cultures of freshC3H responder cells with fresh C57BL/6 DC. No monoclonal antibodies wereadded at this second culture stage. Data for cytokine production thesesecondary cultures are shown in FIG. 17B.

Addition of anti-B7-1 or anti-B7-2 to DC stimulated spleen cultures ledto inhibition of cytokine production (FIG. 16), while in contrastanti-OX-2 monoclonal antibody led an increase in IL-2 production inthese primary cultures (FIG. 16). We have reported similar findingselsewhere (Ragheb et al-submitted for publication). Interestingly,anti-OX-2 abolished the inhibition of cytokine production caused by NPCin these primary cultures (FIG. 17A—see also FIGS. 9, 10 and 13). Inaddition, anti-OX-2 prevented the functional development of a cellpopulation capable of transferring inhibition of cytokine production tofreshly stimulated spleen cells (FIG. 17B).

Discussion

There is considerable theoretical as well as practical interest inunderstanding the mechanism(s) by which a state of antigen specifictolerance can be induced in lymphoid populations. Limits to theeffective induction of tolerance represent a major challenge to moresuccessful allo (and xeno) transplantation, to name but one example(Akatsuka, Y., C. Cerveny, and J. A. Hansen. 1996. Hum. Immunol.48:125-134). Significant efforts have been invested into exploring howpre-(or peri-) transplant donor-specific immunization might produce sucha state (Qian, J. H. et al. 1985. J. Immunol. 134:3656-3663; Kenick, S.,et al. 1987. Transpl. Proc. 19:478-480; Gorczynski, R. M. 1992. Immunol.Lett. 33:67-77; Thelen, M., and U. Wirthmueller. 1994. Curr. Opin.Immunol. 6:106-112; Akolkar, P. N. et al. 1993. J. Immunol. 150 (April1):2761-2773; Ahvazi, B. C. et al. J. Leu. Biol. 58 (1):23-31; Albina,J. E. et al. 1991. J. Immunol. 147:144-152). There is good evidence thatportal venous (pv) immunization somehow leads to tolerance induction,and this immunoregulation can apparently be monitored by followingchanges in cytokine production from host cells, with decreasedproduction of IL-2, IL-12 and IFNγ, and increased IL-4, IL-10, IL-13 andTGFβ (Thelen, M., and U. Wirthmueller. 1994. Curr. Opin. Immunol.6:106-112; Gorczynski, R. M. et al. 1998a. Transplantation. 66:339-349). Which, if any, of these cytokine changes is directly andcausally implicated nevertheless remains obscure.

Further analysis of the cell population able to induce tolerance afterpv immunization led to the somewhat paradoxical observation that donordendritic (DC) cells represented an excellent tolerizing population(Gorczynski, R. M. 1995a. Cell. Immunol. 160:224-231; Gorczynski, R. M.et al. 1996 Transplantation 62:1592-1600). Since antigen-pulsed DC areconventionally thought of as representing an optimal immunizing regime,the mechanism(s) activated following DC pv immunization which led totolerance (Banchereau, J., and R. M. Steinman. 1998. Nature.392:245-252) was of interest. It is already clear that DC themselvesrepresent an extremely heterogeneous population, in terms of origin,cell surface phenotype, turnover in vivo and possibly function (Salomon,B. et al. 1998. J. Immunol. 160:708-717; Leenen, P. J. M. et al. 1998.J. Immunol. 160:2166-2173). In the mouse lymph node at least 3 discretepopulations were identified, one of which comprised small CD8a⁺NLDC145⁺cells, likely of lymphoid origin, with an immature phenotype, and whosenumbers were profoundly increased (100×) following Flt3L treatment invivo (Salomon, B. et al. 1998. J. Immunol. 160:708-717) (administrationof the latter has been reported to lead to proliferation of dendriticcells and other cells of hematopoietic origin (Maraskovsky, E. et al.1996. J. Exptl. Med. 184:1953-1962)). These cells resembled theinterdigitating DC found in the T cell areas of the splenic white pulp,and have been implicated in regulation of immunity induced by other(myeloid derived) DC (Salomon, B. et al. 1998. J. Immunol. 160:708-717;Kronin, V. et al. 1996. J. Immunol. 157:3819-3827; Suss, G., and K.Shortman. 1996. J. Exptl. Med. 183:1789-1796).

A variety of other studies have indicated that the induction of immunity(vs tolerance) following antigen presentation was intrinsicallydependent upon the co-existence of other signaling ligands at thesurface of DC (interacting with appropriate counter-ligands on thesurface of other cells (e.g. stimulated T cells)) (Larsen, C. P. et al.1994. J. Immunol. 152:5208-5219; Lenschow, D. J. et al. 1996. Annu. Rev.Immunol. 14:233-258; Larsen, C. P., and T. C. Pearson. 1997. Curr. Opin.Immunol. 9:641-647). It was speculated that infusion of DC via theportal vein induced tolerance by co-opting another cell population,distinguishable by expression of unique cell surface ligands, whosebiological function was to facilitate induction of tolerance, notimmunity, when antigen was presented in association with otherwiseimmunogenic DC. Some preliminary evidence supporting this hypothesis wasrecently reported (Gorczynski, R. M. et al. 1999. J. Immunol. 162:774-780). Herein, this is referred to as a facilitator cell. Moreover,because pv immunization has been shown to be associated with increasedexpression of a novel molecule, OX-2, previously reported to beexpressed on DC (Barclay, A. N. 1981. Immunology 44:727; Barclay, A. N.,and H. A. Ward. 1982. Eur. J. Biochem. 129:447; Chen, Z. et al. 1997.BBA. Mol. Basis Dis. 1362:6-10; Gorczynski, R. M. et al. 1998b.Transplantation. 65:1106-1114), it was predicted that this moleculewould in fact serve as a “marker” for the hypothetical facilitator celldescribed. Experiments reported herein are consistent with such ahypothesis.

It is here shown that within the hepatic NPC population there is asubset of cells able to inhibit stimulation by allogeneic DC in anon-MHC restricted fashion (see FIGS. 9 and 10), and able to induce thedevelopment of an antigen-specific immunoregulatory cell population invitro (see FIGS. 9 and 10). The non-MHC-restricted nature of this“facilitator” cell interaction indicates that it functions by providingan accessory signal (a regulatory not a co-stimulatory signal) to the DCwhich stimulate T cells in the allogeneic mixed leukocyte reactiondescribed, in a fashion analogous to the original description ofcostimulatory interactions (Jenkins, M. K. et al. 1988. J. Immunol.140:3324-3329). As a result the stimulated lymphocytes alter theircytokine production profile (with decreased IL-2 production andproliferation), and become able to regulate the immune response seenfrom freshly stimulated lymphocytes (see panel B in FIGS. 9 and 10).Most interestingly, following expansion of DC in vivo by Flt3Ltreatment, it is shown that in fact the liver itself contains both animmunostimulating population (large cells by velocity sedimentationanalysis), and this putative “facilitator” cell population (see FIGS.11-15). Furthermore, the latter biological activity resides within aslow-sedimenting (small size) NLDC145⁺ cell population expressingpreferentially both cell surface B7-2 and OX-2 (see FIGS. 11 and 12).When it was investigated whether this same population of cells wasactive in vivo in regulating graft tolerance, it was found again thatafter prior Flt3L treatment, the liver contained a population of cellswhich transferred increased renal graft acceptance (FIG. 15) and inparallel altered the cytokine production profile of immunized micetowards increased IL-4 and TGFβ, and decreased IL-2 and IFNγ production(FIG. 14).

In a final attempt to explore the role for OX-2 expression itself inthis regulatory function, fresh spleen cells were stimulated with DCalone or in the presence of anti-B7-1, anti-B7-2 or anti-OX-2. Note thatother studies (data not shown) have confirmed that even the bone-marrowderived DC used contains small numbers of OX-2⁺ cells (RMG-unpublished).Unlike anti-B7-1 and anti-B7-2 which decreased cytokine production, aresult in keeping with the hypothesized role for these as costimulatormolecules (Hancock, W. W. et al. 1996. Proc. Natl. Acad. Sci. USA.93:13967-13972; Freeman, G. J. et al. 1995. Immunity. 2:523-532;Kuchroo, V. K. et al. 1995. Cell. 80:707-718), anti-OX-2 produced asmall but significant (1.7-2.5 fold in three studies) increase in IL-2production in this system (FIG. 16). Most important, however, inclusionof anti-OX-2 Mab in a system where exogenous “facilitator” cells wereadded (from NPC), blocked completely the induction of inhibitionnormally seen in such cultures (FIGS. 9 and 10; compare with lower panelof FIG. 17). These data are consistent with the concept that OX-2delivers a regulatory, not a costimulatory, signal in this situation.

How does the present data fit within the evolving framework ofunderstanding in the heterogeneity of DC? As noted above, there has beenspeculation that a separate population of CD8a⁺NLDC145⁺ DC of lymphoidorigin which proliferates in response to Flt3L, might be responsible forimmunoregulation. Other data have implicated IL-10 as a cytokine whichmight modify development/maturation of DC into a population expressingincreased amounts of B7-2 and capable of inducing tolerance (Steinbrink,K. et al. 1997. J Immunol. 159:4772-4780). The role of regulation ofexpression of Fas as a controlling feature in this regard is unexplored(Suss, G., and K. Shortman. 1996. J. Exptl. Med. 183:1789-1796). Thedata disclosed herein is the first to implicate another molecule, OX-2,in the delivery of a tolerizing signal, perhaps in association withalterations in expression of B7-2, Fas etc. It is intriguing that whilethere is clearly a key role for intra-thymic DC in the regulation ofself-tolerance (Banchereau, J., and R. M. Steinman. 1998. Nature.392:245-252), natural expression of OX-2 was initially first describedon thymic DC (as well as within the brain) (Barclay, A. N. 1981.Immunology 44:727)-there is as yet no evidence to suggest that thisrepresents a functionally relevant expression for OX-2 in this location.However, other independent data have also implied an immunoregulatoryrole for OX-2 expression, again as assayed by altered cytokineproduction in vitro from cells stimulated in the presence/absence ofexpressed OX-2 (Borriello, F. et al. 1997. J. Immuno. 158:4548).

It has been reported that following pv immunization there is ameasureable expansion in numbers of populations of γδTCR⁺ cells capableof adoptive transfer of increased graft survival to naïve recipients(Gorczynski, R. M. et al. 1996c. Immunology. 87 (3):381-389). Little isknown concerning the nature of the antigen recognized by these cells,and why, as a population, their numbers are preferentially increasedfollowing pv immunization. It is speculated that this may be explainableultimately in terms of a differential susceptibility of γδTCR⁺ vs αβTCR⁺cells to immunoregulatory signals delivered following OX-2 expression.Indeed, FITC-OX-2:Fc bound to >80% of activated γδ T cells and <20% ofactivated αβ T cells.

In conclusion, the inventor has reported for the first time thatfunctional heterogeneity in the DC pool may be understandable in termsof differential expression of OX-2 on the cell surface. Expression ofthis molecule seems to give cells the capability to induceimmunoregulation, increased renal graft survival (and altered cytokineproduction both in vivo and in vitro). The present invention suggeststhat such OX-2 expressing cells are referred to as “facilitator” cells(for tolerance induction).

Example 4 Preparation of Murine Antibodies

Mouse and rat hybridomas to a 43 Kd molecule expressed in the thymus, ona subpopulation of dendritic cells, and in the brain, in mammaliantissue derived from mouse, rat and human were prepared. Using CHO cellstransiently transfected with adenovirus vector(s) expressing a cDNAconstruct for the relevant OX-2 gene, the monoclonal antibodies (Mabs)detect a molecule encoded by this construct (rat OX-2 (rOX-2), mouseOX-2 (mOX-2) and human OX-2 (huOX-2) respectively). Furthermore, atleast some of the anti-rat Mabs detect determinants expressed on themurine OX-2 molecule.

Materials and Methods

Antigen preparation from tissues and Western blotting were performed asdescribed in Gorczynski et al., Transplantation, 1998, 65:1106-1114:

Spleen cells (human samples were obtained from cadavers at the time oforgan retrieval for transplantation) were used for preparation ofdendritic cells/macrophages. Tissue was digested with a mixture ofcollagenase and dispase and centrifuged over lymphopaque. Cells wereadhered for 2 hr at 37° C., washed vigorously, and incubated for 14 hrat 37° C. Dendritic cells were isolated as non-adherent cells(Gorczynski et al., Transplantation, 1996. 62:1592-1600). Routinestaining of mouse splenocytes with NLDC-145 and FITC anti-rat IgG, orFITC-MAC-1 before and after overnight incubation produced the followingstaining pattern in these adherent cells: 8%±2%, 90%±11% and 92%±9%,9%±3% respectively. The crude (non-adherent) dendritic cell preparationwas extracted with lysis buffer, titred to a protein concentration of 10mg/ml, and used for immunization. Some of the same material was usedsubsequently in screening ELISAs (below).

When brain tissue was used in Western gel analysis, whole tissue extractwas electrophoresed in 12% SDS-PAGE and transferred to PVDF membranes(Novex Co., San Diego, Calif.). Putative anti-OX-2 Mabs were used astest reagent, with isotypic antibodies (negative in ELISA tests) used ascontrols. Membranes were developed using either anti-rat or anti-mousehorse radish peroxidase and appropriate substrate.

Immunization and Production of Mabs:

Four female BALB/c mice were initially immunized by intraperitonealinjections with 1 mg of human or rat dendritic antigen in CompleteFreund's Adjuvant. Three subsequent boosts were administered as above,spaced at 3 week intervals, with Incomplete Freundis Adjuvant. When theserum titre had risen more than 10-fold from a pre-immune serum sample,as determined by ELISA, the 2 highest responders were boostedintravenously. Three days later the donor mice were sacrificed and thespleen cells were harvested and pooled. Fusion of the splenocytes withX63-Ag8.6.5.3 BALB/c parental myeloma cells was performed as previouslydescribed (Kohler, G. and C. Milstein. 1975. Nature. 25: p. 256-259),except that one-step selection and cloning of the hybridomas wasperformed in 0.8% methylcellulose medium (Immuno-Precise AntibodiesLtd., Victoria, BC). This proprietary semi-solid medium allows HATselection and cloning in a single step and eliminates the overgrowth ofslower growing desirable clones by faster growing, perhaps undesirable,hybridomas. Clones were picked and resuspended in wells of 96-welltissue culture plates in 200 ml of D-MEM medium containing 1%hypoxanthine/thymidine, 20% Fetal Bovine serum, 1% OPI, and 1×10⁶/mlBALB/c thymocytes. After 4 days, the supernatants were screened by ELISAfor antibody activity on plates coated with the immunizing antigen.Putative positive hybridomas were re-cloned by limited dilution cloningto ensure monoclonality and screened in FACS on extracts prepared frombrain tissue (below).

For the production of rat mAbs, 2 Fisher rats were immunized as abovewith mouse antigen. Essentially the same procedure was followed, exceptthe parental cell line used for the fusion was YB2/0.

ELISA and FACS Analysis of Putative Mabs:

ELISA assays used polystyrene plates pre-coated with 100 ng/mlpoly-L-lysine, followed by overnight incubation with the crude dendriticcell antigen (used for immunization) at 10 mg/ml. Wells were developedafter binding of hybridoma superntatants using the anti-rat/anti-mousehorse radish peroxidase antibodies above and plates were analysed in anautomatic ELISA plate reader (TiterTek Multiskan, MCC/340, FlowLabs,Mississauga, Ontario, Canada).

FACS analysis was performed using putative anti-OX-2 Mabs and thefollowing cells. Fresh peripheral blood leucocytes (PBL), isolated overrat/mouse lymphopaque (Cedarlane laboratories) or Ficoll-Hypaque(human); fresh spleen dendritic cells (isolated after adherence andovernight incubation, as above); and CHO cells transduced with viralvectors engineered to contain a single copy of a cDNA inserted into thenot1/bamH1 sites, encoding the relevant species-specific OX-2, as perpublished sequences (Chen, Z. et al. 1997. BBA. Mol. Basis Dis.1362:6-10; McCaughan, G. W., et al. 1987. Immunogenetics. 25: p.133-135), or with control vector alone. FITC anti-mouse (or anti-rat)IgG was used as secondary antibody.

Mixed Leucocyte Reactivity (MLR) and Cytokine Production:

Allogeneic MLR cultures, using 1:1 mixtures of 2.5×10⁶ responder PBL andmitomycin C treated stimulator PBL, were set up in 24-well cultureplates in 1 ml of aMEM medium supplemented with 10% FCS. Cells wereobtained from C3H responder mice (with stimulator C57BL/6), Lewis (LEW)rats (with Brown Norway, BN, as stimulator), and individual humandonors. Culture supernatants were harvested at 60 hrs and tested fordifferent cytokines using previously described ELISA assays (mouse), orusing CTLL-2 as bioassay for IL-2 production from all responder cellsources (Gorczynski, R. M., et al. 1998c. Immunology. 93: p. 221-229).

Results

Evaluation of a Number of Mabs for Staining of Cell Populations in FreshPBL or Spleen:

All Mabs tested in the experiments herein described were previouslyscreened as described in the Materials and Methods above, and detected amolecule in Western gel of brain extracts with Molecular Weight 42-45Kd, and also stained CHO transduced by OX-2 encoding viral vectors. Datain Table 3 show FACS analysis for these Mabs using fresh cells. The dataare summed over several independent analyses, using a number of Mabsdirected to rat, mouse or human OX-2, for staining of cells harvestedfrom fresh PBL or spleen (adherent cells only were tested for thelatter: these represented some 5%-8% of the total cell population in allcases).

It is clear from Table 3 that PBL in all species tested contained some1.3%-2.5% OX-2⁺ cells by FACS analysis, and that spleen adherent cellssimilarly contained 4%-8% OX-2⁺ cells. As confirmation of the inventor'sprevious work, spleen adherent cells taken from C3H mice or LEW ratstreated 4 days earlier by portal venous immunization with 20×10⁶ (or50×10⁶ respectively) of C57BL/6 (or BN) bone marrow cells showed some3.5-5 fold elevation in OX-2⁺ cells (see Table 3). Under theseconditions specific increases in survival of subsequentallo-transplanted cells/tissue have been reported (Gorczynski, R. M. etal. 1996a. Transplantation 62:1592-1600).

Ability of Anti-OX-2 Mabs to Modulate Cytokine Production in MLR InVitro:

In a final study the issue of whether these Mabs can modify the immuneresponse (as assayed by cytokine production) of cells stimulated in anallogeneic mixed leucocyte reaction (MLR) in vitro was addressed. Theinventor has previously shown that cells taken from mice pretreated byportal allogeneic immunization produce predominantly type-2 cytokines,and that an anti-OX-2 Mab could apparently reverse this polarization incytokine production (and indeed abolish the increased graft survivalseen in such mice). Data in Table 4 confirm these results using 3independent Mabs to mouse OX-2. Further, rat or human cells stimulatedin the presence of anti-rat (or human) OX-2, similarly show morepronounced IL-2 production than cells stimulated in the presence ofisotypic control Ig (or no Ig), without a generalized increase incytokine production (as analysed here by no change in IL-6 production inany group).

Discussion

In the data in this example it is confirmed that using species specificMabs, to human, rat or mouse OX-2, that Mabs to the molecule detected onthe surface of host dendritic cells may play a role in regulatingcytokine production after allostimulation in vitro, and moreparticularly that functionally blocking OX-2 expression leads toenhanced IL-2 production (a type-1 cytokine) after allostimulation(Table 4). Borriello et al also recently reported that OX-2 expressionwas not a costimulator for induction of IL-2 and IFNγ synthesis(Borriello, F. et al. 1997. J. Immuno. 158:4548)-our data imply it is infact a negative signal for type-1 cytokine production. In micepreimmunized by the portal vein, as reported earlier, there is a 4-foldincrease in OX-2 expressing cells in PBL and spleen, and a reversal ofpolarization in cytokine production (from type-2 cytokines to type-1cytokines) after stimulation of cells in the presence of OX-2 (seeTables 3 and 4) (Gorczynski, R. M. et al. 1998b. Transplantation.65:1106-1114).

Example 5 Preparation of Rat Antibodies

Five rats were immunized using GERBU adjuvant (GERBU Biotechnik,Gaiberg, Germany) with 500 mg of membrane protein purified from themouse dendritic cell (DC) line DC2.4 (a gift from K. Rock, Harvard).Serum from these rats was tested 7 days after the third immunization,and compared with a pre-immunization sample in an ELISA usingplate-bound material of Mol. Wt. 40 Kd-45 Kd eluted from Western blots,and Alk Pase anti-rat Ig. Two rats with high titre antibody werere-immunized and sacrificed 4 days later for fusion of spleen cells withHAT-sensitive Sp2/0 parent cells for preparation of hybridomas.Hybridomas were screened by ELISA (56/960+ve), subcloned, and frozen(−70° C.). For further specificity testing of the anti-OX-2 Mabs willuse CHO cells can be transfected with a pBK eukaryotic expression vector(Stratagene, CA) expressing OX-2. Full length OX-2 cDNA, including theleader sequence, was amplified from DC2.4 cells using sense andantisense primers constructed with Spe1 or Xba1 sites respectively attheir 5′ ends for directional cloning into the vector. A band of theexpected size (849 bp) was obtained on agarose gel electrophoresis. Thesequence of the cloned cDNA was confirmed by sequencing using anautomated DNA sequencer (Chen, Z. and Gorczynski, R. M. 1997. Biochem.Biophys. Acta. 100, in press). CHO cells were transfected byelectroporation (5×106 cells in 0.5 ml were pulsed at 960 MH₂ and 120Vusing a Bio-Rad Gene Pulser (Bio-Rad, Hercules, Calif.), using the fulllength OX-2 expression plasmid along with a plasmid encoding puromycinresistance (100:1 ratio), followed by selection in puromycin (12 mg/mlfor 4 days). Puromycin resistant cells were cloned by limiting dilution.5 CHO transfectant clones have been obtained expressing mRNA for OX-2 asconfirmed by PCR. These clones can be used to screen the putative ratanti-mouse OX-2 Mabs.

(a) FACS Staining of Cells from pv Immunized Mice with Anti-Mouse OX-2

A 4-fold increase in staining of spleen and hepatic NLDC145+ (dendriticcell marker) cells from pv immunized mice with anti-rat OX-290 wasobserved. Spleen and liver tissue of mice at various times (12 hours; 2,7 and 14 days) following pv immunization can be sectioned and stained byimmunohistochemistry, using anti-NLDC145, anti-OX-2 Mabs. Single cellsuspensions from the same tissues can be stained, using 3-colour FACS,with FITC-anti-mouse OX-2, rhodamine-anti-NLDC145, andphycoerythrin-anti-T200 (mouse lymphocyte marker). In all cases (bothFACS and immunohistochemistry) the appropriate irrelevant isotypecontrol antibodies are included. Tissue from control mice receivingrenal grafts alone, or following additional iv immunization, can also beexamined. Detection of NLDC145+(and/or MAC-1+) cells showing increasedexpression of OX-2 is predicted in pv immunized mice only (seeGorczynski, R. M. et al. 1998. J. Immunol. 160, in press). The inventorhas shown DC-associated antigen persists only in animals with survivinggrafts (Gorczynski, R. M., Chen, Z., Zeng, H. and Fu, X. M. 1998.Transplantation 66: 339-349). It was also assessed whether anti-OX-2,infused at different times post transplantation, causes rejection (b).

(b) Modulation of Graft Rejection and Cytokine Production by Anti-MouseOX-2

C3H mice receive pv immunization with cultured C57BL/6 bone-marrowderived dendritic cells (DC), CsA and renal allografts. Groups of micereceive intravenous infusion of various rat anti-mouse OX-2 Mabs(100-500 mg/mouse, ×5, at 2 day intervals), beginning at different timespost transplantation (this will be guided by data from (a)). Serumcreatinine and animal survival are followed. Serum from Mab-treated miceare tested in ELISA and by FACS with OX-2 expressing CHO transfectants(above) to ensure antibody excess. If OX-2 expression is important forpv induced increased graft survival, the anti-OX-2 treated pv immunizedmice will reject grafts like untreated controls, with similarpolarization of cytokine production to type-1 cytokines (assayed by PCR;ELISA with cultured, restimulated cells). As controls pv immunized,grafted mice receive anti-CD28 and anti-CTLA4 these Mabs do not modifythe effects of pv immunization as assayed by graft survival orpolarization in cytokine production. It is expected that OX-2 treatmentbut not other Mabs, will simultaneously abolish expansion of γδTCR+cells after pv immunization.

Example 6 Preparation of a Fusion Protein Linking the ExtracellularDomain of OX-2 to Mouse Fc

Immunoadhesins, in which a hybrid molecule is created at the cDNA levelby fusing the extracellular domain (ED) of an adhesion molecule with thecarboxyl terminus of IgG heavy chain, the whole being expressed inmammalian cells or in a baculovirus system, have been powerful tools inthe identification and isolation of the counter ligands for the adhesionmolecule of interest. Ligands for a number of members of the TNFRfamily, were identified in this fashion (Goodwin, R. G. et al. 1993.Eur. J. Immunol. 23, 2631-2641; Gruss, H. and Dower, S. 1995. Blood 85,3378-3404). Interest has developed in the potential application ofimmunoadhesins as therapeutic agents. A CTLA4 immunoadhesion, with thecapacity to bind both B7-1 and B7-2, has been used to inhibit T cellcostimulation and decrease rejection (Larsen, C. P. et al. 1996. Nature381, 434-438). Note that CD28/CTLA4 are not counter ligands for OX-2.The fusion protein, is predicted to alter cytokine production (increasedIL-4, IL-10; decreased IL-2, IFNγ) and increase renal graft survivallike pv immunization. We expect that synergistic blockade ofcostimulation (e.g. by CTLA4-Fc) and triggering of a coregulatorypathway (by OX-2ED-Fc) will induce tolerance and produce indefinitegraft survival.

a) Construction of an OX-2 Fusion Protein with Murine IgGFc2a

A cDNA encoding the extracellular region of OX-2 was amplified by PCR,using a 5′ oligonucleotide primer which inserts a Sal1 site 5′immediately at the start of the V-region sequence and a 3′ primer whichcreates a BamH1 site at the 3′ end (the site of junction with Fc). UsingcDNA prepared from mouse ConA activated spleen cells, with a 5′primercontaining an Spe1 site, and a 3′ primer containing a Sal1 site, thesignal peptide for IL-6 (SP-IL-6) was amplified by PCR and ligated tothe OX-2 amplicon. In frame ligation across the junction of SP-IL-6 andOX-2 was checked by manual sequencing-the final cDNA amplified by the5′SP-IL-6 primer and the 3′OX-2 primer was, as expected, 695 bp. Aplasmid expressing murine IgGFc2a (Fcg2a), modified to create a uniqueBamH1 site spanning the first codon of the hinge region, and with aunique Xba1 site 3′ to the termination codon, has been obtained from Dr.Terry Strom (Zheng, X. X. et al. 1995. Journal of Immunology. 154,5590-5600). The IgGFc2a in this insert has been further modified toreplace the C1q binding motif (rendering it non-lytic) and inactivatethe FcgR1 binding site (see Zheng, X. X. et al. 1995. Journal ofImmunology. 154, 5590-5600). Ligation of OX-2 and IgGFc2a in the correctreading frame at the BamH1 site yields a 1446 bp long open reading frameencoding a single 478-amino acid polypeptide (including the 24-aminoacid IL-6 signal peptide). The homodimer has a predicted 105 kDa Mol Wt,exclusive of glycosylation. The fusion gene is then cloned as anSpe1-Xba1 cassette into the eukaryotic expression plasmid pBK/CMV(Stratagene, CA). This plasmid has a CMV promoter/enhancer and aneomycin-resistance gene for selection using G418. The appropriategenetic construction of the OX-2-Fc can be confirmed by directsequencing after cloning into the plasmid vector (Chen, Z. andGorczynski, R. M. 1997. Biochem. Biophys. Acta. 100, in press)-see alsoabove. The plasmid is transfected into CHO cells by electroporation (seeabove), and selected in medium with 1.5 mg/ml G418 (Geneticin: LifeTechnologies, Inc.). After subcloning, high producing clones areselected by screening culture supernatants in ELISA using anti-OX-2 Mabsas capture antibody, and Alk Pase coupled anti-IgGFc2a as detectionantibody. OX-2-Fc fusion protein is purified from culture supernatantsusing protein A-Sepharose affinity chromatography, dialysed against PBS,filter-sterilized and stored in aliquots at −20° C. The size, and OX-2(+IgGFc2a) specificity of the secreted product can be confirmed usingWestern blot analysis under reducing (+DTT) and non-reducing (−DTT)conditions, with Mabs to OX-2 and rat monoclonal anti-mouse IgGFc2a(Pharmingen). The product can be titrated as an inhibitor for FACSstaining of OX-2 expressing CHO cells (see above) using rat Mabs to OX-2as probe. As a prelude to studies (below) using OX-2-Fc in vivo, thehalf-life (t½) in mouse serum following injection of groups of 6 8-weekC3H mice will be studied. This is carried out by subjecting mice to ivinjections of 50 mg or 10 mg of OX-2-Fc, and obtains serial 50 ml bloodsamples at 0.3, 1, 6, 24, 48, 72 and 96 hours. The serum is analyzed inELISA using plates coated with anti-OX-2 as capture antibody, and AlkPase coupled monoclonal anti-IgGFc2a for detection (thus ensuring theassay detects only OX-2-Fc, not OX-2 or IgGFc2a alone). Based on earlierdata in which Fc fusion proteins were used to extend the in vivohalf-life, a t½ in the range of 30-40 hrs (Zheng, X. et al. 1995.Journal of Immunology. 154, 5590-5600) is predicted.

b) OX-2: IgGFc Immunoadhesion Inhibits MLR

CHO cells were transduced with a vector carry the OX-2:Fc cDNA insert.Supernatant was harvested from the CHO cells at 7 days and was culturedwith 5×10⁶ LEW spleen and 2.5×10⁶ irradiated LBNFI spleen cells. Thesupernatant contained 50 ng/ml OX-2:Fc.

The results, shown in Table 5, demonstrate that the soluble OX-2:Fcimmunoadhesion inhibits IL-2 production and generation of cytotoxic Tcells and induces IL-4 production. These results support the use of OX-2as an immunosuppressant.

c) Use of OX-2:Fc In Vivo for Prevention of Graft Rejection

It was shown in (b) that incubation in the presence of 50 ng/ml OX-2:Fccan inhibit an in vitro MLR reaction. To detect inhibition of in vivograft rejection, C3H mice received C57BL/6 skin grafts along with ivinjection of OX-2:Fc (50 mg/mouse) every 2 days×4 injections. Graftswere inspected daily after 10 days for rejection. In a separate study 3mice/group (receiving saline or OX-2:Fc) were sacrificed at 10 days andspleen cells restimulated in vitro (×48 hrs) for analysis of cytokineproduction. Data for these studies is shown in Tables 6 and 7. It isclear from these data that OX-2:Fc has the potential for use as animmunosuppressant to prolong graft acceptance. Furthermore, inassociation with increased graft survival in this model, OX-2:Fc alterspolarization in cytokine production, as already described for portalvein donor-specific immunization.

Example 7 OX-2 Expression in Placenta

Using in situ hybridization, the inventor has shown that OX-2 is notexpressed in the placenta of mice with increased potential for fetalloss. In contrast, OX-2 is expressed in the placenta of normal,non-aborting mice.

CBA/J and DBA/2J mice were used. Matings of CBA/J (females) with DBA/2Jmales show a high incidence of fetal loss (>80%), unlike the reversescenario. Placental tissue was obtained from matings at 8-11 days ofgestation. Uteri were snap frozen, 5 mm sections cut, and stained with abiotinylated anti-sense probe for murine OX-2. Data shown in FIGS. 18Aand 18B indicate increased expression of OX-2 mRNA (in situ labeling) inthe non-aborting strain combination, with essentially absent expressionin the aborting combination. These data are consistent with the notionthat OX-2 expression prevents spontaneous fetal loss syndrome.

The data show that there are fewer OX-2+ implantation sites on day 8.5of pregnancy in mice which are predisposed to fetal loss syndrome(CBA×DBA/2 matings) by contrast to CBA×BALB/c matings which are not sopredisposed. Fgl2 is the trigger for low, and where OX-2 is alsoexpressed, these potentially doomed implantations are “rescued”. Thisfollows from the finding that the abortion rate is lower than expectedfrom % fgl2++ implantation sites, unless anti-OX-2 mAb is administered.In the latter instance, the abortion rate rises to equate with theestimated proportion of flg2++ implant sites.

Example 8 Treatment of Autoimmune Disease

Collagen-induced arthritis (CIA) is an animal model of rheumatoidarthritis which can be induced in susceptible mice (and rats) followingimmunization at the base of the tail with collagen in Freund's adjuvant.It is associated with activation of IFNγ (type-1 cytokine) producing Tcells, though in humans production of TNFα has also been documented tobe important in the pathophysiology of disease. Given our evidence thatCD200Fc (OX-2:Fc) could inhibit type-1 cytokine production followingallosensitization, we have now investigated the effect of CD200Fc onserum TNFα and IFNγ levels, and on cytokine production by lymphocytestaken from mice and stimulated in vitro with rat collagen. Theexperiments described below are from studies addressing the role ofCD200Fc in suppressing the onset of arthritis, and show clearly thatsusceptible mice immunized with bovine type-II collagen develop asignificant arthritis (˜70% of mice by 30 days post injection), withincreased IFNγ/TNFα production, both of which are abolished by treatmentwith CD200Fc from the time of collagen administration.

Materials and Methods

Animals:

8-week old DBA/1 mice (H2^(q/q)) were obtained from the JacksonLaboratories, Bar Harbour, Me., and kept 5/cage with food and water adlibitum. Mice were entered into experiments at 9 weeks of age.

Collagen Induced Arthritis Model:

Groups of 21 mice received 100 μg bovine collagen type-II (SigmaChemical Co., St. Louis, Mo.) emulsified in Complete Freund's Adjuvant(CFA) in 100 μl 0.1M acetate, injected intradermally at the base of thetail. 18 days later animals received a further injection of 100 μgemulsified in Incomplete Freund's Adjuvant (ICFA) intradermally.Individual animals were monitored daily for signs of arthropathy, whichwas scored independently by 2 investigators for each limb on a 4-pointscale as follows:

0=No paw swelling

1=Paw+a single joint

2=Paw+multiple joints (of the same limb)

3=Joint rigidity

The total score/mouse was recorded (maximum=12). The correlation betweentotal scores for investigators >0.93; scores recorded were thusarithmetic means of the 2 scores for each animal.

Where mice received CD200Fc, 15 μg/mouse was infused iv in 200 μl PBS at3 day intervals into 7 mice/group, beginning on the first day ofcollagen immunization. Control (Hoek et al. (2000)) mice received 15 μgof normal mouse immunoglobulin (pooled from the serum of five 8-weeknormal DBA/1 mice). A further group (7 mice) received no additionaltreatment.

Antigen Stimulation In Vitro and Cytokine Assays:

Pooled peripheral lymph node (PLN) cells (axillary and inguinal nodes),as well as spleen cells, were harvested from individual mice at 36 daysfollowing the first immunization with collagen. Blood was also collectedfrom individual animals by cardiac puncture, and serum obtainedfollowing centrifugation (14,000 rpm for 15 min) after overnightincubation at 4° C. Spleen and PLN cells were cultured in triplicate inmicrotitre plates (1×10⁶ cells/well in 300 μl of α-Minimal EssentialMedium with 10% fetal calf serum, αF10) in the presence or absence of500 ng/ml bovine collagen type-II. A further set of control cultures wasstimulated with 5×10⁵/well mitomycin-C treated (100 μg/ml, 45 min at 37°C.) BALB/c allogeneic spleen stimulator cells.

Culture supernatants (150 μl) were harvested at 40 hrs of culture andassessed for TNFα and IFNγ levels using an ELISA assay and commercialcytokine-specific mAbs obtained from Pharmingen (San Diego, Calif.).Plates precoated with 100 ng/ml R4-6A2 and developed with biotinylatedXMG1.2 were used to assay IFNγ, and those precoated with G281-2626 anddeveloped with biotinylated MP6-XT3 were used to assay TNFα.Streptavidin-coupled alkaline phosphatase with appropriate substrate wasused to develop the assay, and recombinant mouse cytokines (Endogen, SanDiego, Calif.) were used to quantitate the assay.

In addition to analyzing cytokines, proliferation of collagen (andallo)-stimulated cells was assessed by addition of 1 μCi ³HTdR at 72 hrsto each microtitre well (see above), and harvesting the wells forcounting in a well-type β-counter at 14 hrs. All assays were performedin triplicate, with cultures containing unstimulated responder cellsonly serving as an appropriate control.

Measurement of Serum Anti-Collagen Type-1 Antibody Isotypes:

Serum anti-collagen type-II levels were determined by ELISA, usingplates precoated with 100 μl of 1 g/ml collagen type-II and serial3-fold dilutions of the sera (obtained at 36 days post immunization asdescribed above) under test. Plates were developed with isotype-specificbiotinylated rat mAbs to mouse IgM, IgG1, IgG2a/2b and IgG3, followed bystreptavidin-alkaline phosphatase and use of appropriate substrate. AllELISA reagents were purchased from Cedarlane Labs, Hornby, Ontario,Canada. Purified mAb were used to standardize assays in order tocalculate antibody levels. Total IgG levels were determined afteradherence of serum to plates coated with poly-L-lysine (100 ng/ml), anddeveloping with polyspecific biotinylated rat anti-mouse IgG.

Statistical Analysis:

Arthritic scores in different groups were compared by Mann-WhitneyU-tests. Proliferation in, and cytokine production from, cells of allgroups were initially analysed for differences by ANOVA. Thereafterpair-wise comparison of different groups was performed by Student'st-test.

Results

Infusion of Murine CD200Fc Prevents Development of Collagen-InducedArthritis

In order to examine the effect of CD200Fc fusion protein onimmunomodulation of collagen-induced arthritis in mice the followingstudy was performed. Groups of 7 mice were immunized with collagenintradermally, followed by booster immunization 18 days later. Animalsreceived iv injections of either PBS, or 15 μg/mouse of normal mouse Igor CD200Fc, beginning on the day of initial immunization and at 3 dayintervals thereafter. Arthritic scores were assessed for all limbs asdescribed in the Materials and Methods. Data in FIG. 19 are taken fromone of 2 studies of this type, and show the mean (±SEM) for each of the3 groups. These data document that treatment with CD200Fc profoundlyinhibits development of arthritis in this model (p<0.02, Mann WhitneyU-test).

Inhibition of Sensitization to Collagen in CD200Fc-Treated Mice

Data in FIG. 20 show the proliferation in PLN taken from the individualmice shown in FIG. 19, after restimulation of cells for 72 hrs in vitrowith collagen or BALB/c spleen cells. Note that in each of the groupswith high mean arthritic scores (see FIG. 19) 2/7 animals had scores ≦5,within 2SD of the mean of the CD200Fc treated group. Accordingly dataare shown separately for the group of mice with scores ≧6 (5/7 ) and forthose with scores ≦5 (2/7 ).

It is clear from these data that treatment with CD200Fc inhibitssensitization to collagen as assessed by subsequent proliferation inresponse to collagen stimulation (p<0.05 with respect to all othergroups). That this effect is limited to the response initiated at thetime of CD200Fc infusion is clear from the lack of effect onproliferation in response to BALB/c cells. Interestingly, all animalsimmunized in the absence of CD200Fc, even those which did not developovert arthritis (2/7 in each of the PBS injected or control Ig injectedgroups), showed equivalent proliferation in response to collagenrestimulation (see Figure). Thus the expression of disease can beregulated at a stage beyond the sensitization of lymphocytes toautoantigen. Similar data to the above were seen using spleniclymphocytes from these donors (not shown), and for PLN in a separaterepeat experiment.

Inhibition of Serum TNFα and IFNγ in Collagen Sensitized Mice byInjection of CD200Fc

At the time of sacrifice of mice shown in FIG. 20 serum was collectedfor assay of the cytokines TNFα and IFNγ, which have been implicated inthe inflammatory processes responsible for pathophysiology in arthritis.Data shown in FIG. 21 indicate that CD200Fc injection prevents theincrease in serum TNFα and IFNγ levels which are seen in mice with overtarthritis (scores ≧6). Similar data were obtained in a repeat study.Interestingly, unlike the proliferation data in FIG. 20, mice sensitizedin the absence of CD200Fc do not show increased serum cytokine levels ifthey do not simultaneously have arthritic disease. This is consistentwith a causal role for these cytokines in at least some of thesemanifestations of arthritis in this model.

Inhibition of Production of TNFα and IFNγ in Cells of CollagenSensitized Mice by Injection of CD200Fc

Data shown in FIG. 22 are analogous to those of FIG. 21 except that inthis case cytokine production was assayed in the supernatant of culturesfrom the individual mice (whose proliferative capacity was detailed inFIG. 20). Once again injection of CD200Fc clearly led to inhibition ofTNFα and IFNγ production from restimulated cells, in a collagen-specificfashion (no inhibition if restimulation was with BALB/c cells).Interestingly, cytokine production by PLN from mice with/without overtjoint inflammation was equivalent (in the absence of CD200Fc). ThusCD200Fc presumably inhibits sensitization to collagen antigen(s), whilethe presence of collagen-sensitized cells is not alone sufficient fordevelopment of arthritis. Similar data to the above were seen usingsupernatants from splenic lymphocytes from these donors (not shown), andfor supernatants derived from PLN cells in a separate repeat experiment.

Inhibition of Anti-Collagen Antibody Production Following Injection ofCD200Fc

Data shown in FIG. 23 are derived from one of two studies analyzing theisotype profile of anti-collagen antibodies in day 36 sera from the 3groups of mice (control, control-Ig treated and CD200Fc treated). Therewas no difference in total IgG levels in the different groups (data notshown: mean levels 11.2±3.2 mg/ml). It is clear that not only didtreatment with CD200Fc decrease total anti-collagen IgG levels (by˜50%), but there was a clear shift in isotype profiles, withsignificantly more IgG2b relative to all other isotypes produced, and nosignificant inhibition of IgG3 isotype (relative to control mice), inmice treated with CD200Fc.

Discussion

Rheumatoid arthritis is a common chronic inflammatory arthropathy, withsubstantial human cost (Pincus et al. (1986)). Current drug therapy isrelatively non-specific, and has limited efficacy (Scott et al. (1987)).Given the growing evidence that therapies aimed at controlling levels ofinflammatory mediators, which ultimately are causally implicated in thedestruction of cartilage and bone, can ameliorate disease, concertedefforts are directed at developing more of such novel reagents (Choy etal. (2001)). In humans, where TNFα is believed to be critical in thepathophysiology of disease, decoy TNFα receptor molecules, or mAbs toTNFα have both been used successfully for treatment (Moreland et al.(1999); Maini et al. (1999)). Interestingly, IL-10 and IL-4 (both type-2cytokines) which would be expected to diminish inflammatory(predominantly type-1 cytokine driven) responses have not as yetproduced dramatic effects in these clinical settings, perhaps due totheir short half-lives (Choy et al. (2001)).

In mice, in which CIA following immunization with type-II collagen haslong been used as a model of rheumatoid arthritis (Courtenay et al.(1980)), IFNγ has been documented to be crucial for development ofdisease (Boissier et al. (1995)). In keeping with these data, reductionin IL-12 production (believed to be crucial for production of IFNγ invivo) is also associated with amelioration of CIA (McIntyre et al.(1996)). Disease outcome in both wild-type and IFNγ knockout miceimmunized with collagen is more highly correlated with delayed-typehypersensitivity immune reactivity (type-1 cytokines) to autoantigenthan with the presence of circulating autoantibody (Matthys et al.(2000)), data which has been interpreted in terms of a greaterimportance for DTH immunity than antibody in disease pathogenesis.Perhaps somewhat surprisingly, IFNγ-KO mice have a greater incidence ofdisease and more pronounced DTH responses than their wild-typecounterparts, while serum autoantibody levels are suppressed some 5-6fold. These studies and others have contributed to the hypothesis thatin some situations IFNγ may play a protective role in CIA (Matthys etal. (2000); Nakajima et al. (1990); Ortmann et al. (2001)). RecentlyOrtmann and Shevach (Ortmann et al. (2001)) speculated that IFNγ mightact in a protective manner early in disease development, while worseningpathology later in disease. They also reported a protective effect ofIL-10 on disease severity, while IL-4, somewhat paradoxically, increaseddisease, perhaps by augmenting levels of anti-collagen Ig. Matthys etal. (2001) have concluded that IFNγ may exert its primary role inregulating disease severity by modulating development of Mac-1⁺ cellswhose proliferation is induced by mycobacterial products used in theFreund's Adjuvant commonly used in CIA models.

In the inventors' previously reported transplant models, the inventorshad found that increased cellular expression of the molecule CD200 wasassociated with improved survival of allografts, in association withspecific suppression of allo-specific CTL and type-1 cytokine production(IL-2, IFNγ) with sparing or augmentation of IL-10 and IL-4 (Gorczynskiet al. (1998); Gorczynski et al. (1999)). The inventors speculated thatCD200 might play a general immunosuppressive role which could be ofvalue in autoimmune disorders (Chen et al. (1997); Gorczynski et al.(1998)). The same immunosuppressive effect was also achieved by infusionof the solubilized form of CD200, CD200Fc (Gorczynski et al. (1999)),and the inventors accordingly investigated in the studies describedabove whether CD200Fc was effective in decreasing induction of CIA inmice. Our data show unequivocally that this is indeed the case (see FIG.19). Moreover, the cytokine changes observed, both in serum levels ofIFNγ and TNFα (FIG. 21), and in production of the same cytokines afterstimulation in vitro (FIG. 22), are in general accord with the previousreports in this murine model (Boissier et al. (1995); McIntyre et al.(1996)). That the early infusion of CD200Fc actually blockedsensitization of collagen-reactive cells was inferred both from thecytokine data of FIG. 22 and the data of FIG. 20, showing that PLN ofthese mice even failed to proliferate in response to collagenstimulation. In keeping with earlier data studying the effect of CD200Fcon an anti-sheep erythrocyte antibody response, CD200Fc did indeedinhibit (by ˜50%) the development of specific autoantibody to collagenin treated mice (see FIG. 23), along with causing a significant shift inthe isotype profile of the Ig produced typical of that seen in more Th2driven responses (relatively more IgG2b and IgG3 and less IgG2a).Whether the changes in autoantibody, and indeed of cytokine production,represent epiphenomena, or are causally linked with the reduction inarthritis in these mice, remains to be analysed in detail.

While no measures of IL-4 or IL-10 were performed, previous work withCD200Fc in allosensitized mice would lead us to predict that bothcytokines would be elevated in CD200Fc and collagen-injected mice(Gorczynski et al. (1999); Gorczynski et al. (2000)). IL-10 might thenproduce the arthritis-protective effect suggested by others (Ortmann etal. (2001)). Interestingly, sensitization of T cells for increased IFNγand TNFα production alone does not explain CIA in this model. Theinventors observed uniform cytokine production from PLN in these mice(FIG. 22), but only ˜70% of animals produced elevated serum cytokinesand developed overt joint disease (FIGS. 19 and 21). It is interestingto note that while CD200 itself has a relatively ubiquitous expressionpattern (Barclay (1981)), expression of CD200R is more limited. Both ourown group (Gorczynski et al. (2000)) and others (Hoek et al. (2000))have documented existence of CD200R⁺ cells in a macrophage/myeloidpopulation, and the inventors have also suggested expression of CD200Rexists on subpopulations of T cells (Gorczynski et al. (2000)). Theimportant site(s) at which CD200R is expressed in the CIA modeldescribed, and where CD200Fc might thus be presumed to have its mostimportant effect, has not yet been unequivocally identified.

In summary, the results are consistent with a crucial role for infusedCD200Fc in regulation of induction of CIA in mice. Consequently CD200may be useful in treating and preventing autoimmune disease.

Example 9 Cancer Therapy

Materials and Methods

-   Mice: Male C3H/HeJ, BALB/c and C57BL/6 mice were purchased from the    Jackson laboratories, Bar Harbour, Me. Mice were housed 5/cage and    allowed food and water ad libitum. All mice were used at 8-12 weeks    of age.-   Monoclonal antibodies: The following monoclonal antibodies (mAbs)    were obtained from Pharmingen (San Diego, Calif., USA) unless stated    otherwise: anti-IL-2 (S4B6, ATCC; biotinylated JES6-5H4); anti-IL-4    (11B11, ATCC; biotinylated BVD6-24G2); anti-IFNγ (R4-6A2, ATCC;    biotinylated XMG1.2); anti-IL-10 (JES5-2A5; biotinylated, SXC-1);    anti-IL-6 (MP5-20F3; biotinylated MP5-32C11); anti-TNFα (G281-2626;    biotinylated MP6-XT3); FITC anti-CD80, FITC anti-CD86 and FITC    anti-CD40 were obtained from Cedarlane Labs, Hornby, Ontario. The    hybridoma producing DEC205 (anti-mouse dendritic cells) was a kind    gift from Dr. R. Steinman, and was directly labeled with FITC. FITC    anti-H2K^(b), FITC anti-H2K^(k), and anti-thy1.2 monoclonal    antibodies (mAbs) were obtained from Cedarlane Labs, Hornby,    Ontario. Unconjugated and PE-conjugated rat anti-mouse CD200 was    obtained from BioSpark Inc., Mississauga, Ontario, Canada (Ragheb et    al. 1999). CD200Fc was prepared in a Baculovirus expression system,    using a cDNA encoding a murine IgG2aFc region (a kind gift from Dr.    T Strom, Harvard, USA) which carried mutations to delete complement    binding and FcR sites, as we described elsewhere (Gorczynski et al.    1999). Rat monoclonal antibody to CD200^(r) was prepared from rats    immunized with CHO cells transfected to express a cDNA encoding    CD200^(r) (Gorczynski 2001). Anti-CD4 (GK1.5, rat IgG2b) and    anti-CD8 (2.43, rat IgG2b) were both obtained from ATCC, and used    for in vivo depletion by iv infusion of 100 μg Ig/mouse weekly. A    control IgG2b antibody (R35.38), as well as strepavidin horse radish    peroxidase and recombinant mouse GM-CSF, was purchased from    Pharmingen (San Diego, Calif.).    Preparation of Cells: Single Cell Spleen Suspensions were Prepared    Aseptically and after centrifugation cells were resuspended in    α-Minimal Essential Medium supplemented with 2-mercaptoethanol and    10% fetal calf serum (αF10). CD200^(r+) LPS splenic Mph, stained    (>20%) with FITC-CD200Fc, were obtained by velocity sedimentation of    cells cultured for 48 hrs with 1 mg/ml LPS (Gorczynski et al. 2000).    Bone marrow cells were flushed from the femurs of donor mice, washed    and resuspended in αF10. Cells were depleted of mature T lymphocytes    using anti-thy1.2 and rabbit complement.

C1498 (a spontaneous myeloid tumor) and EL4 (a radiation induced thymomatumor) cells were obtained from The American Type Culture Collection(ATCC, Rockville, Md.). Cells used for transplantation into mice werepassaged weekly (5×10⁶ cells/mouse) intraperitoneally in stock 8-weekold C57BL/6 recipients. For experimental tumor challenge either 5×10⁶EL4 tumor cells, or 5×10⁵ C1498 cells, were given intraperitoneally togroups of 6 mice (see results)-animals were sacrificed when they becamemoribund. EL4 cells stably transfected to express CD80 or CD86 wereobtained from Dr. J. Allison, Cancer Research Labs, UC Berkeley, Calif.,while C1498 transfected with CD80/CD86 (cloned into pBK vectors) wereproduced in the author's laboratory. Tumor cells (parent andtransfected) were stored at −80° C. and thawed and cultured prior touse. Cells used for immunization, including the tumor cells transfectedwith CD80/CD86, were maintained in culture in aMEM medium supplementedwith 10% FCS. Untransfected and transfected cells of each tumor linewere used for immunization within 2 passages in culture. Over this timein culture transfected cells repeatedly showed stable expression (byFACS) of CD80/CD86 (>80% positive for each tumor assayed over a 6 monthperiod with multiple vials thawed and cultured). Non-transfected tumorcells did not stain with these mAbs (<2%).

CD200^(r+) cells were obtained from lymphocyte-depleted murine spleencells. Cells were treated with rabbit anti-mouse lymphocyte serum andcomplement (both obtained from Cedarlane Labs. Hornby, Ontario),cultured with LPS (10 μg/ml) for 24 hours, and separated intopopulations of different size by velocity sedimentation (Gorczynski etal. 2000). Small CD200^(r+) cells stained >65% by FACS withanti-CD200^(r) antibody (Gorczynski 2001).

Bone marrow transplantation (BMT): C57BL/6 mice received 300 mg/Kgcyclophosphamide iv 24 hrs before intravenous infusion of 20×10⁶T-depleted C3H or C57BL/6 bone marrow cells. Immediately prior to usefor tumor transplantation (28 days following bone marrow engrafting), asample of PBL (50 μl/mouse) was obtained from the tail vein ofindividual mice and analysed by FACS with FITC-anti-H2K^(k) orFITC-anti-H2K^(b) mAb. Cells from normal C57BL/6 or C57BL/6reconstituted C57BL/6 mice were 100% H2K^(b) positive, as expected. Insimilar fashion, PBL from C3H mice were 100% H2K^(k) positive. H2K^(k)positive cells in the C3H-reconstituted C57BL/6 mice by FACS comprised85%±8.5% of the total cell population (mean over ˜100 mice used in thestudies described below). Mice in all groups were gaining weight andhealthy.Cytotoxicity and Cytokine Assays:

In allogeneic mixed leukocyte cultures (MLC) used to assess cytokineproduction or CTL, responder spleen cells were stimulated with equalnumbers of mitomycin-C treated (45 min at 37° C.) spleen stimulatorcells in triplicate in αF10. Supernatants were pooled at 40 hr fromreplicate wells and assayed in triplicate in ELISA assays for lymphokineproduction as follows, using capture and biotinylated detection mAbs asdescribed above. Varying volumes of supernatant were bound in triplicateat 4° C. to plates pre-coated with 100 ng/ml mAb, washed ×3, andbiotinylated detection antibody added. After washing, plates wereincubated with strepavidin-horse radish peroxidase (Cedarlane Labs),developed with appropriate substrate and OD₄₀₅ determined using an ELISAplate reader. Recombinant cytokines for standardization were obtainedfrom Pharmingen (U.S.A.). All assays showed sensitivity in the range 40to 4000 pg/ml. CTL assays were performed at 5 days using cells harvestedfrom the same cultures (as used for cytokine assays). Variouseffector:target ratios were used in 4 hr ⁵¹Cr release tests with 72 hrConA activated spleen cell blasts of stimulator genotype.

Quantitation of CD200 mRNA by PCR:

RNA extraction from spleen tissue of tumor injected mice was performedusing Trizol reagent. The OD280/260 of each sample was measured andreverse transcription performed using oligo (dT) primers (27-7858:Pharmacia, USA). cDNA was diluted to a total volume of 100 μl with waterand frozen at −70° C. until use in PCR reactions with primers for mouseCD200 and GAPDH (Gorczynski et al. 1998). Different amounts of standardcDNA from 24 hr cultures of LPS stimulated peritoneal macrophages (knownto express CD200 and GAPDH) were amplified in six serial 1:10 dilutionsfor 30 cycles by PCR, in the presence of a tracer amount of ³²P. Sampleswere analysed in 12.5% polyacrylamide gels, the amplicons cut from thegel, and radioactivity measured in a β-counter. A standard curve wasdrawn for each set of primer pairs (amplicons). cDNAs from the variousexperimental groups were assayed in similar reactions using 0.11 cDNA,and all groups were normalized to equivalent amounts of GAPDH. CD200cDNA levels in the different experimental groups were then expressedrelative to the cDNA standard (giving a detectable ³²P signal over fivelog₁₀ dilutions). Thus a value of 5 (serial dilutions) indicates a testsample with approximately the same cDNA content as the standard, while avalue of 0 indicates a test sample giving no detectable signal in anundiluted form (<1/10⁵ the cDNA concentration of the standard).

Results

Growth of EL4 or C1498 Tumor Cells in C57BL/6 Mice, and in Allogeneic(C3H) BMT Mice:

Groups of 6 C57BL/6 mice received iv infusion of 300 mg/Kgcyclophosphamide (in 0.5 ml PBS). A control group received PBS only, asdid a control group of 6 C3H mice. 24 hrs later cyclophosphamide treatedC57BL/6 mice received iv injection of 20×10⁶ T-depleted bone marrowcells pooled from C57BL/6 mice (syngeneic transplant), or C3H mice(allogeneic transplantation). All groups of animals receivedintraperitoneal injection (in 0.5 ml PBS) of 5×10⁶ EL4 or 5×10⁵ C1498tumor cells (see FIG. 24) 28 days later. Animals were monitored dailypost tumor inoculation.

Data in FIG. 24 (one of 2 such studies) show clearly that while C3H micerejected both EL4 and C1498 (allogeneic) leukemia cell growth, 100%mortality was seen within 9-12 days in normal C57BL/6 mice, or insyngeneic bone marrow reconstituted mice. Interestingly, despite theabsence of overt GVHD (as defined by weight loss and overall health),two-thirds of C3H reconstituted C57BL/6 mice rejected EL4 tumor cells,reflecting the existence of a graft versus leukemia effect (GVL) (panela of FIG. 24), and there was a marked delay of death for mice inoculatedwith C1498 leukemia cells (panel b of Figure). In separate studiessimilar findings were made using tumor inocula (for EL4/C1498respectively) ranging from 2×10⁶-10×10⁶, or 1.5×10⁵-10×10⁵(RMG-unpublished).

Immunization of Normal C57BL/6 Mice for Protection Against EL4 TumorGrowth:

Blazar and co-workers reported immunization for protection from tumorgrowth in C57BL/6 mice using tumor cells transfected to over-expressmouse CD80 (Blazar et al. 1997). Using CD80 and CD86 transfected EL4cells obtained from this same group, or C1498 cells transfected withCD80/CD86 in our laboratory, we immunized groups of 6 C57BL/6 mice ipwith Complete Freund's adjuvant (CFA) alone, or with CFA mixed with5×10⁶ mitomycin-C treated tumor cells, or CD80/CD86 transfected tumor.Animals received 2 injections at 14 day intervals. 10 days after thelast immunization all mice received 5×10⁶ EL4 tumor cells, or 5×10⁵C1498 cells, and mortality followed. Data are shown in FIG. 25 (1 of 2studies), for EL4 only.

In agreement with a number of other reports, mice pre-immunized withCD80-transfected EL4 survive significantly longer after challenge withviable EL4 tumor cells than non-immunized animals, or those immunizedwith non-transfected cells or CD86 transfected cells (p<0.05)-see alsoFIG. 28. Similar data were obtained using CD80-transfected C1498 cells(RMG-unpublished). In separate studies (not shown) mice immunized withtumor cells in the absence of Freund's Adjuvant failed to show anyprotection from tumor growth. However, equivalent protection (to thatseen using Freund's Adjuvant) was also seen using concomitantimmunization with poly(I:C) (100 μg/mouse) as adjuvant (data not shown).

Role of CD4⁺ and/or CD8⁺ Cells in Modulation of Tumor Growth after BMT.

In order to investigate the effector cells responsible for leukemiagrowth-inhibition in mice transplanted with allogeneic bone marrow (seeFIG. 24), BMT recipients received weekly injections of 100 μg/mouseanti-CD4 (GK1.5) or anti-CD8 (2.43) mAb, followed at 28 days by leukemiacell injection as described for FIG. 24. Depletion of CD4 and CD8 cellsin all mice with these treatments was >98% as defined by FACS analysis(not shown).

As shown in FIG. 26 (data from one of 3 studies), and in agreement withdata reported elsewhere (Blazar et al. 1997), in this BMT model tumorgrowth inhibition for EL4 cells is predominantly a function of CD8rather than CD4 cells, while for C1498 leukemia cells growth inhibitionwas equally, but not completely, inhibited by infusion of eitheranti-CD4 or anti-CD8 mAb (see pane b of Figure).

Evidence that Tumor Rejection in BMT Mice is Regulated by CD200:

Previous studies in rodent transplant models have implicated expressionof a novel molecule, CD200, in the regulation of an immune rejectionresponse. Specifically, blocking functional expression of CD200 by amonoclonal antibody to murine CD200 prevented the increased graftsurvival which followed donor-specific pretransplant immunization, whilea soluble form of CD200 linked to murine IgG Fc (CD200Fc) was a potentimmunosuppressant (Gorczynski et al. 1998; Gorczynski et al. 1999). Inorder to investigate whether expression of CD200 was involved inregulation of tumor immunity, we studied first the effect of infusion ofCD200Fc on suppression of resistance to growth of EL4 or C1498 tumor inBMT mice as described in FIG. 24, and second the effect of CD200Fcinfusion in mice immunized with CD80-transfected tumor cells asdescribed in FIG. 25. Note this CD200FC lacks binding sites for mousecomplement and FcR (see Materials and Methods, and Gorczynski et al.1999). In all cases control groups of mice received infusion ofequivalent amounts of pooled normal mouse IgG. Data for these studies isshown in FIGS. 27 and 28 respectively (data from one of 2 studies ineach case).

It is clear that suppression of growth of either EL4 or C1498 tumorcells in BMT mice is inhibited by infusion of CD200Fc, but not by poolednormal mouse IgG (FIG. 27). CD200Fc also caused increased mortality inEL4 or C1498 injected normal C3H mice. Data in FIG. 28 show thatresistance to EL4 tumor growth in EL4-CD80 immunized mice (as documentedin FIG. 25) is also inhibited by infusion of CD200Fc. In separatestudies (not shown) a similar inhibition of immunity induced by CD80transfected C1498 was demonstrated using CD200Fc.

Effect of Anti-CD200 mAb on Resistance to Tumor Growth in Mice Immunizedwith CD80/CD86 Transfected Tumor Cells:

As further evidence for a role for CD200 expression in tumor immunity inEL4-CD80/EL4-CD86, or C1498-CD80/C1498-CD86 immunized mice, theinventors examined the effect of infusion of an anti-CD200 mAb on EL4 orC1498 tumor growth in this model. Infusion of anti-CD200 into micepreimmunized with EL4-CD86, or C1498-CD86 uncovered evidence forresistance to tumor growth. Separate studies (not shown) revealed thatanti-CD200 produced no significant perturbation of EL4 growth in the EL4or EL4-CD80 immunized mice, or of C1498 growth in C1498 or C1498-CD86immunized mice. These data suggest that immunization with EL4-CD86 orC1498-CD86 elicited an antagonism of tumor immunity resulting fromincreased expression of CD200. Thus blocking the functional increase ofCD200 expression with anti-CD200 reversed the inhibitory effect. Notethat in studies not shown, these same effects of anti-CD200 have beenreproduced (in mice immunized with CD86-transfected tumor cells) withF(ab′)₂ anti-CD200 (RMG-unpublished).

To confirm that indeed immunization with CD86-transfected tumor cellswas associated with increased expression of CD200, the study shown inFIG. 29 was repeated, and sacrificed 3 mice/group at 4 days followingEL4 tumor injection. RNA was isolated from the spleen of all mice, andassayed by quantitative PCR for expression of CD200 (using GAPDH as“housekeeping” mRNA control). Data for this study are shown in FIG. 30,and show convincingly that CD200 mRNA expression was >5-fold increasedfollowing preimmunization with EL4-CD86, a condition associated withincreased tumor growth compared with EL4-immunized mice (FIGS. 25 and29). In dual-staining FACS studies (not shown), with PE-anti-CD200 andFITC-DEC205, the predominant CD200⁺ population seen in control andimmunized mice were DEC205+ (>80%)-see also Gorczynski et al. (1999).Similar results were obtained using C1498 tumor cells (data not shown).

Given this increase in CD200 expression following preimmunization withCD86-transfected cells, and the evidence that CD200 is associated withdelivery of an immunosuppressive signal to antigen encountered at thesame time, the inventors also examined the response of spleen cellstaken from these C57BL/6 mice to allostimulation (with mitomycin-Ctreated BALB/c spleen cells), in the presence/absence of anti-CD200 mAb.Data from one of 3 studies are shown in Table 8. Interestingly, micepreimmunized with EL4-CD86 cells show a decreased ability to generateCTL on alloimmunization with third-party antigen (BALB/c), and decreasedtype-1 cytokine production (IL-2, IFNγ), with some trend to increasedtype-2 cytokines (IL-4 and IL-10). These effects were reversed byinclusion of anti-CD200 in culture, consistent with the hypothesis thatthey result from increased delivery of an immunosuppressive signal viaCD200 in spleen cells obtained from these animals (Gorczynski et al.1998).

Evidence for an Interaction Between CD200 and CD200^(r+) Cells inInhibition of EL4 Tumor Growth:

Inhibition resulting from infusion of CD200Fc into mice follows aninteraction with immunosuppressive CD200^(r+) cells (Gorczynski et al.2000). At least one identifiable functionally active population ofsuppressive CD200^(r+) cells was described as a small, F4/80⁺ cell in apool of splenic cells following LPS stimulation (Gorczynski et al.2000)—F4/80 is a known cell surface marker for tissue macrophages. In afurther study we investigated whether signaling induced byCD200:CD200^(r) interaction (where CD200^(r+) cells were fromlymphocyte-depleted, LPS stimulated, spleen cells) was behind thesuppression of tumor immunity seen following CD200Fc injection. Allgroups of 6 recipient mice received tumor cells ip. In addition toinfusion of CD200Fc as immunosuppressant (in FIG. 31), one group of C3Hreconstituted animals received CD200^(r+) cells (in FIG. 31: >65% ofthese cells stained with an anti-CD200^(r) mAb), while a final groupreceived a mixture of both CD200Fc and CD200^(r+) cells. It is clearfrom the Figure that it is this final group, in which interactionbetween CD200 and CD200^(r) is possible, which showed maximum inhibitionof tumor immunity compared with the C3H reconstituted control mice.

Role of CD4⁺ and/or CD8⁺ Cells in CD200 Regulated Modulation of TumorGrowth after BMT:

Data in FIG. 26 above, and elsewhere (Blazar et al. 1997) show thattumor growth inhibition for EL4 cells is predominantly a function of CD8rather than CD4 cells, while for C1498 leukemia cells growth inhibitionwas equally, but not completely, inhibited by infusion of eitheranti-CD4 or anti-CD8 mAb. To investigate the role of CD200 in theprotection mediated by different T cell subclasses, the followingadditional studies were performed. In the first, C57BL/6 recipients ofC3H BMT received (at 28 days post BMT) inoculations of EL4 or C1498tumor cells along with anti-CD4, anti-CD8 or CD200Fc alone, orcombinations of (anti-CD4+CD200Fc) or (anti-CD8+CD200Fc). Survival wasfollowed as before (see FIG. 32—one of 2 studies). In a second study(FIG. 33—data from one of 2 such experiments) a similar treatmentregimen of mAbs or CD200Fc alone or in combination was used to modifygrowth of EL4 tumor cells in mice preimmunized with EL4-CD80 cells asdescribed earlier in FIG. 25.

Data in panel a of FIG. 32 confirm the effects previously documented inFIGS. 26 and 27, that CD200Fc and anti-CD8 each significantly impairedthe growth inhibition in BMT recipients of EL4 cells, while anti-CD4 mAbwas less effective. Combinations of CD200Fc and either anti-T cell mAbled to even more pronounced inhibition of tumor immunity in the BMTrecipients, to levels seen with non-allogeneic transplanted mice. Datawith C1498 tumor cells (panel b) were somewhat analogous, though as inFIG. 26, anti-CD4 alone produced equivalent suppression of growthinhibition to anti-CD8 with this tumor. As was the case for the EL4tumor, combinations of CD200Fc and either anti-T cell mAb causedessentially complete suppression of C1498 tumor growth inhibition. Bothsets of data, from panels a and b, are consistent with the notion thatCD200Fc blocks (residual) growth-inhibitory functional activity in bothCD4 and CD8 cells, thus further inhibiting tumor immunity remainingafter depletion of T cell subsets.

Data in FIG. 33, using EL4 immunized BL/6 mice, also showed combinationsof CD200Fc and anti-CD4 treatment produced optimal suppression of tumorimmunity to EL4 cells, consistent with an effect of CD200Fc on CD8⁺cells. Anti-CD8 alone abolished tumor immunity in these studies, so anypotential additional effects of CD200Fc on CD4⁺ cells could not beevaluated.

Discussion

In the studies described above, we have asked whether expression of themolecule CD200, previously reported to down-regulate rejection oftissue/organ allografts in rodents (see previous Examples), wasimplicated in immunity to tumor cells in syngeneic hosts. Two modelsystems were used. The one, in which tumor cells are injected into micewhich had received an allogeneic bone marrow transplant followingcyclophosphamide pre-conditioning, has been favoured as a model forstudying potential innovative treatments of leukemia/lymphoma in man(Blazar et al. 1997; Imamura et al. 1996; Blazar et al. 1999; Champlinet al. 1999). In the other EL4 or C1498 tumor cells were infused intoBL/6 mice which had been preimmunized with tumor cells transfected tooverexpress the costimulatory molecules CD80 or CD86. These studies werestimulated by the growing interest in such therapy for immunization ofhuman tumor patients with autologous transfected tumor cells (Imro etal. 1998; Brady et al. 2000; Jung et al. 1999; Freund et al. 2000;MartinFontecha et al. 2000).

In both sets of models we found evidence for inhibition of tumor growth(FIGS. 24 and 25) which could be further modified by treatment designedto regulate expression of CD200. Infusion of CD200Fc suppressed tumorimmunity (led to increased tumor growth, and faster mortality) in bothmodels (FIGS. 27 and 28), while anti-CD200 improved tumor immunity inmice immunized with CD86-transfected EL4 or C1498 tumor cells (FIG. 29).This latter finding suggests that the failure to control tumor growthfollowing immunization with EL4-CD86 or C1498-CD86 was associated withoverexpression of endogenous CD200, a hypothesis which was confirmed byquantitative PCR analysis of tissue taken from such mice (FIG. 30).CD200 was predominantly expressed on DEC205⁺ cells in the spleen ofthese mice (see text), which was associated with a decreased ability ofthese spleen cell populations to respond to allostimulation in vitro(see Table 8). Non antigen-specific inhibition following CD200expression formed the basis of our previous reports that a soluble formof CD200 (CD200Fc) was a potent immunosuppressant (Gorczynski et al.1998). Consistent with the hypothesis that increased expression of CD200in mice immunized with CD86-transfected tumor cells was responsible forthe inhibition of alloreactivity seen in Table 8, suppression wasabolished by addition of anti-CD200 mAb (see lower half of Table 8).Earlier reports have already documented an immunosuppressive effect ofCD200Fc on alloimmune responses (Gorczynski et al. 1999), and productionof antibody in mice following immunization with sheep erythrocytes(Gorczynski et al. 1999).

Maximum inhibition of tumor immunity was achieved by concomitantinfusion of CD200Fc and CD200r+ cells (F4/80⁺macrophages—see FIG. 31).We next investigated the cell type responsible for tumor growthinhibition whose activity was regulated by CD200:CD200^(r) interactions.The data confirmed previous reports that EL4 growth inhibition waspredominantly associated with CD8 immune cells, while immunity to C1498was a function of both CD4+ and CD8+ cells (Blazar et al. 1997). Forboth tumors in BMT models suppression of tumor growth inhibition wasmaximal following combined treatment with CD200Fc and either anti-T cellmAb, consistent with the idea that CD200 suppression acts on both CD4⁺and CD8⁺ T cells.

A number of studies have examined immunity to EL4 or C1498 tumor cellsin similar models to those described above (Blazar et al. 1997; Boyer etal. 1995), concluding that CD8⁺ cells are important in (syngeneic)immunity to each tumor, and CD4⁺ T cells are also important in immunityto C1498 (Blazar et al. 1997). Evidence to date suggests that NK cellmediated killing is not relevant to tumor growth inhibition in BMT miceof the type used above (Blazar et al. 1997). Other reports haveaddressed the issue of the relative efficiency of induction of tumorimmunity in a number of models following transfection with CD80 or CD86,and also concluded that CD80 may be superior in induction of anti-tumorimmunity (Blazar et al. 1997; Chen et al. 1994), while CD86 may lead topreferential induction of type-2 cytokines (Freeman et al. 1995). Thisis of interest given the cytokine production profile seen in EL4-CD86immunized mice (Table 8), which is similar to the profile seen followingCD200Fc treatment of allografted mice (Gorczynski et al. 1998). EL4-CD86immunized mice show increased expression of CD200 (FIG. 30), with noevidence for increased resistance to tumor growth (FIG. 25). Resistanceis seen in these mice following treatment with anti-CD200 (FIG. 29A).Somewhat better protection from tumor growth is seen using viable tumorcells for immunization, rather than mitomycin-C treated cells as above(Blazar et al. 1997). Whether this would improve the degree ofprotection from tumor growth in our model, and/or significantly alterthe role of CD200:CD200^(r) interactions in its regulation, remains tobe seen.

There are few studies exploring the manner in which suppression mediatedby CD200:CD200^(r) interactions occurs. In a recent study in CD200 KOmice Hoek et al observed a profound increase in the presence ofactivated macrophages and/or macrophage-like cells (Hoek et al. 2000),and we and others had previously found that CD200^(r) was expressed onmacrophages (Gorczynski et al. 2000; Wright et al. 2000). The inventorsalso reported that CD200^(r) was present on a subpopulation of T cells,including the majority of activated γδTCR⁺ cells (Gorczynski et al.2000), a result we have recently confirmed by cloning a cDNA forCD200^(r) from such cells (Kai et al—in preparation). γδTCR+ cells maymediate their suppressive function via cytokine production (Gorczynskiet al. 1996), while unpublished data (RMG—in preparation) suggests thatthe CD200^(r+) macrophage cell population may exert its activity viamechanisms involving the indoleamine 2,3-dioxygenase (IDO) tryptophancatabolism pathway (Mellor et al. 1999). We suggest that the mechanismby which CD200Fc leads to suppression of tumor growth inhibition in themodels described is likely to be a function both of the tumor effectorcell population involved (FIGS. 32, 33) as well as the CD200^(r) cellpopulation implicated in suppression.

In a limited series of studies (not shown) we have used other BMTcombinations (B10 congenic mice repopulated with B10.D2, B10.BR or B10.Abone marrow) to show a similar resistance to growth of EL4 or C1498tumor cells, which is abolished by infusion of CD200Fc. Studies are inprogress to examine whether DBA/2 or BALB/c mice can be immunized toresist growth of P815 syngeneic (H2^(d)) tumor cells by P815 cellstransfected with CD80/CD86, and whether this too can be abolished byCD200Fc. Taken together, however, our data are consistent with thehypothesis that the immunomodulation following CD200:CD200^(r)interactions, described initially in a murine allograft model system, isimportant also in rodent models of tumor immunity. This has importantimplications clinically.

Example 10 Trim in the FSL Sarcoma Lung Metastasis Model:Cues fromPregnancy

Allogeneic leukocyte-induced transfusion-related immunomodulation (TRIM)has been shown to enhance tumor growth (Vamvakas et al. 1994; Bordin etal. 1994). OX-2 is expressed on a variety of cells in transfused blood(i.e. a subpopulation of dendritic cells and possibly B cells) (Wrightet al. 2000; Hoek et al. 2000), thus the effect of anti-OX-2 on the TRIMenhancement of FSL10 lung nodules was examined.

Materials and Methods

Enhancement of Lung Nodules by TRIM

A dose response curve demonstrated a plateau in the TRIM enhancement oflung metastases with 50, 100 or 200 μl of BALB/c heparinized blood given4 days after tail vein injection of the cultured tumor cell by tail vein(see FIG. 34). A dose of 200 μl of BALB/c heparinized blood (about15-20% of blood volume) was given 4 days after tail vein injection ofthe cultured tumor cells as a physiologically suitable model in which toscreen for treatments that have a major abrogating effect on TRIM.

All animals were monitored for signs of illness daily, and 21 days aftertumor inoculation, the mice were sacrificed, the lungs were removed andfixed in Bouin's solution, and the number of surface nodules wascounted. To deal with variation in number of metastases between mice,20-25 mice per group were used and medians were calculated (usinglog-transformed data, where 0 nodules was set at 0.1 for that animal).It was then possible to assess the significance of differences in logmean±sem with respect to our a priori hypotheses using Student's t test,and to construct 95% confidence intervals for the medians. Differencesin the proportion of mice in different groups with no visible metastaseswas assessed by the χ² statistic, or by Fisher's Exact test whereappropriate.

FIG. 34A shows the median number of lung nodules in C57Bl/6J micereceiving the indicated dose of freshly-prepared allogeneic BALB/cstrain blood by tail vein. The effect is seen if the blood is given 7days prior to, or 4 days after a tail vein injection of 1×10 6 FSLsarcoma cells. FSL10 is a methlycholanthrene-induced fibrosarcomagenerated in C57Bl/6 mice and maintained by standard tissue culture invitro. Such cells are weakly antigenic. Group size is 20-25 per group,and P values showing increased numbers to lung nodules are on thefigure. FIG. 34B shows the proportion of mice with no tumor nodules. Pvalues were determined by Student's t test for A, and by Chi-square orFisher's Exact test for B.

The Role of Dendritic Cells

MAb to a myeloid DC/APC surface marker (5 μg anti-CD11c) or lymphoiddendritic cells (DC) (5 μg supernatant of DEC205 hybridoma, an amountshown to be sufficient using in vitro assays of DC function (Gorczynskiet al. 2000)) was added to 200 μl of BALB/c blood or to PBS. The TRIMenhancement of tumor growth was analyzed using the same method as above.

Results

Enhancement of Lung Nodules by TRIM

FIG. 35A represents the effect of adding anti-OX-2 monoclonal antibody(3B6, 1 ug per million leukocytes) to the blood (or PBS control) beforetumor cell transfer. A control is the same amount of 3B6 in PBS. Thetotal dose was 3.3 ug per mouse. FIG. 35A shows this amount ofanti-CD200 in PBS had no effect, whereas when added to blood, thestimulation of tumor nodule number was prevented, —indeed, it wasreduced below control levels. FIG. 35B shows % with no lung nodules. Inthis and subsequent studies, 2×10 6 FSL cells were used, and the bloodwas always given 4 days after this.

As illustrated in FIG. 35, the enhancement of lung nodules in mice given2×10⁵ sarcoma cells by 200 μl of BALB/c blood compared to phosphatebuffered saline control (PBS) given 4 days after tumor injection, wascompletely blocked by adding 3.3 μg of anti-OX-2 (3B6 monoclonalantibody (mAb)) to the blood before the transfusion. The mAb in PBS hadno effect. Interestingly, the proportion of mice with lung metastaseswas boosted by allogeneic blood compared to PBS but was reduced by bloodto which anti-OX-2 had been added. The median number of nodules wasgreater in this study in part because we had doubled the tumor cellinoculum, but we do see experiment-to-experiment variation in the numberof nodules in the control group which has been important in executinglarge experiments, as will be discussed.

The Role of Dendritic Cells

FIG. 36A is a repetition of FIG. 35A which confirms the effect ofanti-OX-2, but with addition of antibodies to dentritic cells.Anti-CD11c was used for myeloid dendritic cells, and DEC205 for lymphoiddendritic cells. The latter are usually CD8-positive. It can be readilyappreciated that monoclonal antibody to lymphoid dendritic cells had noeffect on the stimulation of lung metastases, whereas anti-CD11c blockedthe effect. The reason the number of nodules is not below control isthought to be due to the existence of OX-2-positive and OX-2-negativeCD11c-type dendritic cells. The latter stimulate immunity, and this isseen when anti-OX-2 is used to block one of the subsets. Anti-CD11cleads to loss of both subsets.

The results show that anti-CD11c, but not DEC205, abrogated the TRIMeffect (* indicates significant increase over control, ** indicatessignificant abrogation of TRIM, *** indicates significant decrease belowcontrol, P<0.05). There was no effect of anti-OX-2, DEC205 or anti-CD11cin PBS injected as a control (data not shown). Due to the large numberof treatment groups in this experiment, it could not be done in a singleday. Therefore, 5 mice in each group were treated in 4 experiments andthe data was examined and pooled. The result therefore compensates forany effect of day-to-day variation in tumor cells, mice or blood usedfor transfusion.

Example 11 OX-2 Prevents Fetal Loss

Successful pregnancy in allopregnant mice can also be viewed asdependent upon control of graft rejection. Proinflammatory Th1 cytokines(TNF-α+IFN-γ+IL-1) can cause spontaneous abortion in mice by a mechanismwhich involves a novel prothrombinase, fgl2, which promotes fibrindeposition. However, the inventors found that spontaneous abortion ratesin abortion-prone CBA×DBA/2 matings and in low abortion rate CBA×BALB/cmatings were lower than the frequency of implantation sites showingfibrin^(hi)+fgl2 mRNA^(hi). OX2 expression was present in the same sitesas fgl2 mRNA, and neutralization of this OX2 expression by anti-OX-2raised the abortion rate to predicted levels. Conversely, an OX2immunoadhesin dramatically reduced the abortion rate. Therefore, inaddition to its role in organ and tissue allograft rejection, OX2expression is involved in the prevention of spontaneous abortiontriggered by cytokine up-regulation of fgl2 at the feto-maternalinterface.

In the Example detailed below, evidence is shown using anti-OX2monoclonal antibody and a soluble form of OX2 in which the extracellulardomains of the molecule are linked to an Ig Fc region (OX2:Fc)(described in Example 6), that OX2 is fundamentally important toachieving successful allopregnancy.

Methods

All of the techniques used, including mixed leukocyte cultures, cytokineanalysis, and allografting, are detailed in previous publications(Gorczynski et al. 1996a; Arck et al. 1997a; and Clark 1999). Theanti-OX2 mAb (3B6) was obtained from BioCan (Mississauga, Ontario)(Gorczynski et al. 1998b). 100 μg/mouse was used for each injection. Apolyclonal, affinity-purified, rabbit antibody to fgl2 was describedelsewhere, and used ip at a dose of 22 mg/mouse. OX2Fc immunoadhesin wasgiven ip (35 μg/mouse).

Results

In Situ Expression of OX2 mRNA Following Renal Transplantation orAllopregnancy:

OX-2 has been reported at the fetomaternal interface usingimmunohistochemistry in rats (Example 7 and Bukovsky et al. 1984). Todetermine whether OX-2 was expressed in the uterus of allopregnant mice,the inventors carried out in situ hybridization for OX-2 mRNA inCBA×DBA/2 and CBA×BALB/c matings. Adjacent sections of the tissuesamples were also used to stain for fgl2 mRNA (fgl2 is a prothrombinasemolecule up-regulated by certain Th1 cytokines implicated triggeringpregnancy loss). For comparison, we also examined OX2 expression inliver sections from C3H mice receiving C57BL/6 renal allograftsfollowing donor-specific portal vein preimmunization, a treatment whichpromotes tolerance and which is critically dependent upon up-regulationof expression of OX-2 on hepatic APC. Typical patterns for uterinestaining (pregnant mice) are reported elsewhere (Clark et al. 2001),along with cumulative data for OX2 and fgl2 expression in the uteri ofpregnant control mice and mice treated with TNFα+IFNγ to increaseabortion rates (Clark et al. 2001).

In these studies the inventors found that OX2 mRNA expression wasup-regulated following pv immunization and renal transplantation, and inallopregnant mice. In pregnant mice the inventors found a negativecorrelation between expression of the molecules fgl2 and OX-2 which didnot reach statistical significance (Clark et al. 2001). With followingcytokine treatment of pregnant animals, and prior to the onset ofabortions, the proportion of fgl2^(hi) implantations increased, althoughthis also did not achieve significance due to small numbers. However,with cytokine treatment, the proportion of OX-2^(hi) implants decreaseddramatically. These data support the hypothesis that in pregnancy, fgl2and OX-2 expression are reciprocally regulated by cytokines, that theirlevels affect pregnancy outcome, and that a major determinant of successor failure of fgl2^(hi) ‘at risk’ implantations was the presence orabsence of OX2. Most interestingly, the inventors also reported thatcontinued expression of OX2, as occurs in pregnancy, was essential forsuccessful survival of allografts following pv pretransplantimmunization, and for the concomitant changes in cytokine productionseen in those animals (previous Examples and Gorczynski et al. 2000).

Effect of anti-OX2 mAb on Renal Transplant Survival and PregnancyOutcome:

Fifty to 70% of implantations in control and cytokine-boosted CBA×DBA/2pregnancies show the fgl2^(hi) phenotype (Clark et al. 2001). Aninjection of anti-Vg1.1 on day 8.5 of pregnancy, 1 day before abortionsbecome evident, inactivates most of the trophoblast-recognizing γδsubset producing IL-10 and TGFβ and boosts abortion rates toapproximately 48% (Arck et al. 1999). The inventors hypothesized thatthe “suppressor” γδT cells inactivated by this treatment might bedependent upon OX2 expression for their functional activity. Theinventors have reported that following allotransplantation, the kineticsof expression of OX2 follows closely the development of immunoregulatoryγδT cells. Functional blockade of OX2 expression (by anti-OX2 treatment)reverses increased graft survival (see FIG. 2) and prevents adoptivetransfer of tolerance by γδT cells. To test whether OX-2 expression inpregnancy might similarly be activating anti-abortion mechanisms which‘rescued’ fgl2^(hi) implant sites from proceeding to embryo death whereboth OX-2 and fgl-2 were expressed, the inventors injected controlCBA×DBA/2- and CBA×BALB/c-mated mice with the same anti-OX-2 monoclonalantibody that blocks induction of transplantation tolerance. FIG. 37shows that injection on or after day 8.5 increased the spontaneousabortion rate to that expected if all fgl2hi sites in CBA×DBA/2proceeded to resorb. The increase in abortion rate was not due to atoxic effect of anti-OX-2 on the embryo because co-administration ofanti-fgl2 to neutralize prothrombinase activity abrogated the boost inabortion rates (data not shown). Injection of anti-OX-2 intoCBA×BALB/c-mated mice also increased the abortion rate to 22%,consistent with the 21% fgl2 mRNA^(hi) implantation site frequency.

Infusion of OX2 Immunoadhesin Modulates Renal Allograft Rejection andSpontaneous Abortion:

As further “proof of principle” that OX2 expression is functionallyimportant for increased allograft survival the inventors used animmunoadhesin (OX2:Fc), in which the extracellular domains of OX2 werelinked to a murine IgGFc region, to investigate modulation of allograftrejection and pregnancy. Data in the previous Examples has alreadyindicated that this molecule has potent immunoregulatory properties invivo, including the ability to decrease allograft rejection. Data inFIG. 38 represent a cumulative comparison of the effect of infusion ofOX-2:Fc on renal allograft survival or rate of abortions in CBA×DBA/2mated mice. Once again there was a clear parallel between the functionalactivity of OX2:Fc measured in these two assays.

Discussion

The notion that the rejection of organ allografts would be mimickedimmunologically by immune recognition of the fetus in allopregnantmothers has been with us for decades. There has been intense interest inthe role of altered cells and soluble factors (e.g. cytokines) in thephenomena seen in both situations. As an example, with decreased graftrejection (and successful pregnancy) there are numerous reports of thepresence of unique γδT cells with “suppressor phenotypes”, and alteredcytokine patterns, with elevated levels of type-2 cytokines, inparticular IL-10 and TGFβ (Chaouat et al. 1999; Tsuda et al. 2001; Clarket al. 2001b; Arck et al. 1999 and Arck et al. 1997a). In contrast,allograft rejection in rodents and man has been associated with elevatedtype-1 cytokines, and previous studies have also shown that both TNFαand IFNγ must be present for spontaneous abortions to be induced(Chaouat et al. 1999; Tsuda et al. 2001; Clark et al. 2001b; Arck et al.1999 and Arck et al. 1997a). Since the fgl2 gene is activated by IFNγbut not by TNFα, the inventors have hypothesized that the obligatoryrole for the latter cytokine either involves activation of PMNLessential for abortions to be completed, or down-regulates OX-2expression.

It is worthy of note that in the inventors' previous work, and in thestudies described above, expression of fgl2, a thrombosis-inducingmolecule, was up-regulated on the trophoblast in response to cytokines.Cytokine-treated IRF1^(−/−) females mated to +/+ males do not abort, asthere is no up-regulation of fgl2 in the maternal decidual tissues andthe best explanation for lack of abortions is that fgl2⁺ trophoblast andfgl2⁺ maternal decidua must meet. In the regions where the two tissuesmeet, a zone of spontaneous cleavage, enough enzymatic activitypresumably occurs to cause necrosis. The inventors have also documenteda basal level of fgl2 expression in trophoblast tissue. Given theevidence that excessive anticoagulation with heparin or hirudin leads toretroplacental hemorrhage fatal to the embryo and sometimes mother, theinventors suggest that this low (basal) level of expression of themolecule fgl2 may reflect a normal homeostatic role for fgl2 inpreventing spontaneous bleeding. However, cytokine-mediated (by TNFα andIFNγ) upregulation of fgl2 is associated with increased rates ofabortion. These effects can in turn be counteracted by the combinationof both TGF-β and IL-10, both perhaps produced by trophoblast cells,which are known to inhibit cell-mediated vascular injury and clotting.There is little data to date examining the role of fgl2 prothrombinasein allograft rejection. Interestingly xenograft rejection, a process inwhich acute and subacute vascular changes are believed crucial, isreportedly less pronounced in an fgl2 knockout mouse (Levy et al,personal communication). It thus becomes extremely interesting to knowwhether fgl2 has a more general role in immunomodulation in both alloand fetal grafts.

The present data shed further light on the mechanisms by which some ofthese changes occur in both the allopregnant mouse and inallo-transplant, by providing evidence for a crucial role for alteredexpression of another molecule OX2, in regulating both embryo executiontriggered by cytokine up-regulation of fgl2 prothrombinase and themodulation of renal allograft rejection. The inventors have proposedthat OX2 acts in transplantation as a co-stimulatory signal thatdeviates cytokine production away from Th1 (e.g. IL-2, IFN-γ) andtowards Th2/3 (e.g. IL-4, IL-10, TGF-β) production. Associated with thisis expansion of a γδT cell subset that mediates tolerance viasuppression. In support of such a hypothesis, the inventors have shownincreased expression of OX2 is associated with decreased rejection andaltered cytokine production; that the kinetics of expression of OX2parallels altered cytokine production and γδT cell expansion; and thatthese effects are diminished by infusion of anti-OX2 mAbs, and enhancedby infusion of the immunoadhesin OX2:Fc. The inventors have now alsodocumented that a similar correlation exists between OX2 expression andfetal loss in allopregnant mice, even when abortion rates are increasedfollowing infusion of cytokines (where our data suggests OX2 continuesto act to counter the effects of fgl2).

It is now known that OX2 functions following interaction with itsreceptor (OX2^(r)) on target cells. At least 2 groups, ourselves(Gorczynski et al. 2000 and Wright et al. 2000) have documented theexistence of OX2^(r) on macrophages, and the inventors showed optimalinhibition of graft rejection in vivo occurred with infusion of bothOX2:Fc and OX2r+ cells (Gorczynski et al. 2000). Unlike the Barclaygroup, the inventors have found that a large percentage (>80%) of ConAactivated γδT cells also express an OX2^(r), as defined by FACS withFITC-OX2:Fc. The inventors have recently confirmed this independentlyusing our mAbs to OX2r, and following cDNA sequencing of the OX2rexpressed in γδTCR+ hybridomas (Yu et al: manuscript in preparation).The inventors have not yet studied the functional activity of OX2r cells(whether macrophages or γδT cells) in allopregnant mice. However,extrapolating from the data shown above, it is suggested that increasedexpression of OX2^(r) will correlate with successful allopregnancy, andthat triggering intracellular signaling by cross-linking OX2r on cellsby mAb (presumably in the same fashion as native cell-bound OX2 doeswhen it interacts with OX2^(r)) rescues putatively doomed embryos micefrom cytokine-induced spontaneous abortion (mediated by elevated fgl2expression).

Example 12 OX-2 Expression Rescues Putatively Doomed Embryos

The importance of CD200 (OX-2) in rescuing potentially doomed mouseembryos is illustrated by the in situ hybridization result in FIG. 39.The pattern of hybridization with anti-sense probe for fgl2 (top panels)is compared with the pattern of hybridization using anti-sense probe forOX-2 for CBA×DBA/2 mated mice on day 8.5 of gestation. (Sense controlsshow no staining). Details of methods are provided in Molecular HumanReproduction 2001 (Clark et al. 2001). In section 1, left, fgl2 is ++and OX-2 essentially negative. In section 2, fgl2 is still ++ but inthis implantation, OX-2 hybridization can be seen in the same areas asfgl2. In section 3, fgl2 is only weakly expressed, and OX-2 is readilydemonstrable. It has been shown that the observed rate of abortions isless than expected from the % of implantations showing fgl2⁺⁺. Whenanti-OX-2 neutralizing antibody is administered, all of the fgl2⁺⁺implantation appear to be aborted. This is attributed to the demise ofimplantations showing pattern #2.

Example 13 OX-2 is Expressed in Successful Human Pregnancy

The importance of OX-2 (CD200) is rescue of potentially doomed fgl2^(hi)mouse implantations has been shown above. Increased fgl2 expression isalso seen in abortion of chromosomally normal human embryos in the firsttrimester, but not in chromosomally abnormal embryos. To determine ifOX-2 is expressed in successful pregnancies (for which first trimestertissue is not available), term placentae were examined. The placentaewere obtained after delivery, the decidua was scraped off, and thetrophoblast cells were isolated by sequential enzymatic digestion, asdescribed by Guilbert et al. To remove non-trophoblast cells, anti-CD9antibody was added, and the suspension was passed over ananti-immunoglobulin column, according to the method of Guilbert et al.The adhered CD9⁺ ‘stroma’ was obtained by physical agitation of thebeads of the column. RNA was extracted in the usual manner from asimilar number of CD9⁺ and CD9⁻ cells and RT-PCR was performed (30cycles) using primers rto the full-length (3 exon) OX-2 gene, usingprimers to the 3rd exon that detects the truncated (exon 2-missing)mRNA. The PAGE gel in FIG. 40 shows the molecular size ladder (lane 1),trophoblast with full length primers (lane 2) and exon 3 primers (lane3). The full length and shorter mRNA OX-2 transcripts are seen. Lane 4(corresponding to lane 2) and lane 5 (corresponding to lane 3) representnegative result obtained with CD9⁺ stromal cells.

To test for OX-2 protein expression on trophoblast, the flow cytometryprotocol (summarized at the end) was used with the CD9⁻ fraction.Surface expression of OX-2 was detected using PE-tagged monoclonalantibody to human OX-2 (Cedarlane labs). Intracellular cytokeratin wasstained using FITC-mAB from Dako. FIG. 41 shows result using antibodydetecting CK5, 6, 8, 17; similar results have been obtained usingFITC-anti-CK18. The upper left panel shows the forward and side scatterpattern. FITC and PE isotype controls used to set gates is shown belowon left. Upper right panel shows cytokeratin-positive cells, themajority of which were also OX-2⁺. A significant population of CK-OX-2⁺⁺ cells was also noted. The identity of this population has notbeen established. On the right, the middle panel shows staining profilefor OX-2 for the CK- and CK⁺ populations, and the lower panel shows thescatter. Clearly these populations have different properties. Velocitysedimentation separation indicates both populations may haveOX-2-dependent immunoregulatory activity.

Example 14 OX-2 Mab in the Prevention of Postcoital Pregnancy

The inventors have shown that infusion of an immunoadhesin, OX-2:Fc,reduces the rate of abortion in a mouse model system using animals withincreased rates of spontaneous abortion. Antibodies to OX-2 on the otherhand were shown to increase the rates of spontaneous abortion, anddecrease litter size in this model. This allows the manipulation offertility using non-hormonal methods.

This example confirms the ability of OX-2 Mab to reduce fertility inrodents.

The objection of this experiment is to determine if OX-2 Mab is capableof preventing or reducing the fertility in rodents after coitus.

Reduction in fertility is defined as:

-   a) the percent of treated impregnated females delivering pups is    less than that observed in the control group.-   b) the average litter size of treated impregnated females carrying a    litter to term is less than that observed in the control group.    Materials and Methods

Rodents (rats/mice) were housed in individual cages with one female andone male/cage. Animals were inspected daily for vaginal plugs. Beginningon the day of fertilization (defined as the presence of a vaginal plug)females were kept one to a cage, and received ip injections of mAbs toOX2 rat anti-mouse (clone 3B6) for mice and mouse anti-rat (clone 6C2)for rats. The control group of the impregnated females receivedinjections with control rat or mouse Ig.

Records were kept of the number of impregnated females producing littersin each group, as well as the litter size for each mother.

Animals

-   Mouse female, male—outbred Swiss-Webster.-   Rat female, male—outbred Sprague-Dawley.    Statistical Considerations

Comparison of the percent of impregnated females carrying litters toterm, and the mean litter size for successful pregnancies, were madeindependently for rats/mice using non parametric Chi-square or Fisher'sExact test where appropriate for pregnancy rates, and the Rank Sum testfor litter sizes.

Methodology

Breeding

One animal of each sex is placed in a cage. The females are checkeddaily for vaginal plugs. Once a vaginal plug was observed the femaleswere placed one to a cage.

Beginning on the day of impregnation (defined as the day a vaginal plugis observed), each female was moved to a new cage (housed alone) andbegins treatment with mouse or rat OX2 antibodies by intraperitonealinjection.

Experiment No. 1

-   Mice: dose 100 micrograms per mouse every 3 days for 7 doses.-   Rats: dose 300 micrograms per rat every 3 days for 7 doses    Experiment No. 2-   Mice: dose 250 micrograms per mouse every 36 hours for up to 18 days-   Rats: dose 500 micrograms per rat every 3 days for 7 doses    Methods of Assessment-   Assessment of coitus: vaginal plug.-   Assessment of pregnancy—number of pups delivered-   In the second experiment the sex was also determined in each pup    delivered.-   The number of animals giving birth is counted as are the number of    pups delivered.-   Each female animal is housed in an individual cage until day 25    after breeding. Animals not delivering by day 25 were considered not    to be pregnant and destroyed.    Study End Points-   Number of animals in each group who delivered any pups.-   Number of pups delivered to each animal achieving delivery at term.    Duration of the Study-   Length of time the animal were held post impregnation . . . 25 days.-   Methods of termination . . . pups were sacrificed after recording    the total litter size per female. In the second study the sex of    each pup was also recorded.-   Tissue to be taken . . . none.    Schedule of Evaluations-   Daily inspection of animals for impregnation (vaginal plugging)-   Injection of pregnant animals every 3 days in the rat experiments    and experiment 1 for the mice. The mice in experiment two were    observed every 36 hours.-   Daily inspection from day 19 for evidence of birthing.    Test Article and Concomitant Treatments    Test Article-   Anti-OX2 mAbs were obtained from BioSpark, Canada.-   Mouse anti-rat OX2: clone 6C2. Cat #SP 300X. Lot #02

Stored at 1 mg/ml (no azide) in PBS.

-   Rat anti-mouse OX2: clone 3B6. Cat #SP 200X Lot #03

Stored at 1 mg/ml, no azide, in PBS.

-   Control Ig was also obtained from BioSpark, Canada. This represents    a preparation from pooled normal ascites (from rats or mice).-   Again storage is in PBS, with no azide, at 1 mg/ml.-   Normal rat Ig: Lot #01. Cat. #SP89-   Normal mouse Ig: Lot #01; Cat #SP99-   Inventory and accountability records are kept by the investigator-   a) The investigator kept study drugs in a locked freezer, a secure    storage facility, in his laboratory at the TGH (MBRW-2-926).    Experiment 1:    Mice:-   Control: 17/17 mice impregnated had successful pregnancies.-   Mean litter size: 7-   3B6 injected: 17/18 mice impregnated had successful pregnancies.-   Mean litter size: 6-   See Table 9-   Statistics: Mice: 17 control and 18 treated-   The proportion of mice pregnant was not significantly different by    Fisher's Exact test; the litter size in each group, and the average    number of pups per group were not significantly different by the    Rank Sum test.    Rats:-   Control: 11/12 rats impregnated had successful pregnancies.-   Mean litter size: 9-   6C2 injected: 5/8 rats impregnated had successful pregnancies.-   Mean litter size for the group was 5 but for the 5 rats delivering    pups the mean litter size was 8-   See Table 11-   P<0.05 for successful pregnancies.-   Statistics: Rats 12 control and 8 treated-   The proportion of animals pregnant in the treated group was not    significantly reduced (P=0.153, Fisher's Exact test). Litter size in    those delivering was reduced (P<0.018, Rank Sum test), and the total    number of pups generated by the immune treated group was reduced    (P<0.006, Rank Sum test)    Experiment 2:    Mice:-   Control: 8/8 mice impregnated had successful pregnancies.-   Mean litter size: 6-   3B6 injected: 3/8 mice impregnated had successful pregnancies.-   Mean litter size for the group was 2 but for the 3 rats delivering    pups the mean litter size was 5-   See Table 10-   Statistics: Mice 8 control and 8 treated-   The proportion of rats pregnant was significantly reduced by    treatment (P=0.0128, Fisher's Exact test), and the number of pups    overall was reduced (P<0.00021, Rank Sum test). The average litter    size for animals successfully delivering was also reduced (P<0.024,    Rank Sum test), but the number of litters was small.    Rats:-   Control: 6/6 rats impregnated had successful pregnancies.-   Mean litter size: 10-   6C2 injected: 2/6 rats impregnated had successful pregnancies.-   Mean litter size for the group was 2.5 but for the two rats    delivering pups the mean litter size was 7.5-   See Table 12-   Statistics: Rats 6 control 6 treated-   The proportion of animals that delivered litters was significantly    reduced by treatment (P=0.03, Fisher's Exact test), as was the total    number of pups produced by the treated group (P<0.002, Rank Sum    test). Litter size was also slightly reduced in those delivering    (P<0.03, Rank Sum test), but the number of litters was small.

CONCLUSIONS

The administration of OX-2 antibodies to rats and mice followingimpregnations has been shown to reduce the number of animals deliveringpups. The average liter size of antibody treated animals that diddeliver was not different from the control animals.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

TABLE 1 Summary of sequences and clones detected in cDNA library from pvimmunized mice Number of clones Match category represented (%) Knownmouse genes 30 (45) Non-mouse genes (rat/human) 10 14 (21) No data basematch 22 (34) Footnotes: Genes were considered a “match” with a BLASTscore >250 with a minimum of 50 bp alignment.

TABLE 2 Cytokine production from cells of mice receiving pv immunizationand anti-rat OX-2 Mabs given to Cytokine levels in culturesupernatants^(b) recipients^(a) IL-2 IFNγ IL-4 IL-10 No pv immunization(CsA only) None 750 ± 125  85 ± 18 29 ± 8 130 ± 40  +anti-rat OX-2 890 ±160  93 ± 19  30 ± 10 120 ± 35  +anti- 415 ± 88*  57 ± 9*  105 ± 22* 275± 55* mouse CD28 +anti-  505 ± 125* 65 ± 8  95 ± 20* 190 ± 45 mouseCTLA4 +anti-B7-1 340 ± 65*  35 ± 7*  120 ± 21* 285 ± 60* +anti-B7-2495 ± 90* 64 ± 7  90 ± 20* 185 ± 45  PV Immunization + CsA None 190 ±55  25 ± 8 107 ± 21 780 ± 150 +anti-rat OX-2  730 ± 140*  60 ± 16*  33 ±10* 220 ± 40* +anti- 145 ± 38  20 ± 9 145 ± 34 1140 ± 245  mouse CD28+anti- 85 ± 25 15 ± 6 125 ± 31 960 ± 220 mouseCTLA4 +anti-B7-1 110 ± 30 20 ± 6 144 ± 28 885 ± 180 +anti-B7-2 75 ± 20 14 ± 5 150 ± 30 1230 ± 245 Footnotes: ^(a)3 C3H mice/group were used in each experiment. Allanimals received CsA and C57BL/6 renal transplants as described in theMaterials and Methods. Mice in the lower half of the Table also receivedpv infusions of 15 × 10⁶ C57BL/6 bone marrow derived dendritic cells onthe day of transplantation. Where monoclonal antibodies were given thedose used was 100 mg/mouse, ×4 doses at 2 day intervals. All mice weresacrificed 14 days post transplantation. Spleen cells were cultured intriplicate from individual animals for 40 hrs in a 1:1 mixture withirradiated C57BL/6 spleen stimulator cells. ^(b)Arithmetic mean (±SD)for triplicate determinations from individual samples of the animalsdescribed in the first column. All cytokines were assayed by ELISA.IL-2, IL-4 and IL-10 are shown as pg/ml, IFNγ as ng/ml. Data are pooledfrom 2 such studies (total of 6 individual mice tested/group).*represents significantly different from control group with no Mab (p <0.02)

TABLE 3 FACS staining of PBL and spleen adherent cells in differentspecies, using anti-OX-2 Mabs Donor^(b) Percent stained cells^(c)SPECIES^(a) Treatment Mab PBL Spleen Human NONE H4B4 1.5 ± 0.3 4.8 ± 1.7H4A9A2 1.5 ± 0.4 6.1 ± 2.0 H4A9C7 1.3 ± 0.4 4.3 ± 1.7 Mouse NONE M3B51.9 ± 0.4 6.7 ± 2.1 M3B6 1.7 ± 0.4 5.2 ± 1.6 M2C8 1.4 ± 0.4 4.2 ± 1.4Mouse PV immune M3B5 5.9 ± 1.5  20 ± 4.1 M3B6 5.2 ± 1.4  17 ± 3.6 M2C84.7 ± 1.4  15 ± 3.3 Rat NONE RC6A3 1.3 ± 0.3 5.3 ± 1.6 RC6C2 1.5 ± 0.46.5 ± 1.7 RC6D1 1.9 ± 0.6 6.8 ± 1.5 Rat PV immune RC6A3 4.8 ± 1.3  16 ±4.2 RC6C2 4.9 ± 1.6  18 ± 3.9 RC6D1 5.3 ± 1.7  20 ± 4.5 Footnotes:^(a)Fresh cells were obtained from normal human donors (PBL), cadaverictransplant donors (human spleen), or from adult (8-10 week) mouse or ratdonors. The same 3 separate tissue donors were used for each Mab tested.^(b)Donor pretreatment refers to infusion of allogeneic bone marrowcells into the portal vein (C57BL/6 for C3H mouse donors; BN for LEW ratdonors) 4 days before harvest of PBL or spleen (see text and (6)).^(c)Arithmetic mean (+SD) for percent cells stained in 3 independentassays. Control antibodies (FITC anti-mouse IgG (for anti-human oranti-rat Mabs, or FITC anti-rat IgG for anti-mouse Mabs) gave nosignificant staining above background (<0.2%).

TABLE 4 Type-1 cytokine production in MLR cultures is increased byanti-OX-2 Mabs Cytokine levels in culture supernatants^(b) ELISA assays(murine only) Bioassay (CTTL-2) Mabs in culture^(a) IL-2 IFNγ IL-4 IL-10IL-2 IL-6 MOUSE MLR None 350 ± 55  35 ± 18 345 ± 63 340 ± 50 480 ± 160365 ± 74 M3B5 890 ± 160* 115 ± 29* 130 ± 10* 168 ± 42* 820 ± 200* 265 ±46 M3B6 915 ± 155* 117 ± 25* 135 ± 32* 135 ± 38* 850 ± 175* 303 ± 55M2C8 855 ± 155* 105 ± 28* 120 ± 32* 140 ± 37* 830 ± 165* 279 ± 61control Ig 370 ± 75  36 ± 11 330 ± 55 310 ± 45 335 ± 60 349 ± 59 None**710 ± 145 108 ± 23 110 ± 21 105 ± 23 690 ± 155 285 ± 54 RAT MLR None 490± 145 360 ± 57 RC6A3 690 ± 155* 295 ± 55 RC6C2 845 ± 180* 345 ± 68 RC6D1830 ± 160* 370 ± 57 Control Ig 475 ± 160 356 ± 58 HUMAN MLR None 395 ±85 295 ± 45 H4B4 570 ± 125* 315 ± 50 H4A9A2 630 ± 145* 320 ± 48 H4A9C7625 ± 140* 345 ± 56 Control Ig 360 ± 120 320 ± 50 Footnotes: ^(a)MLRcultures were set up as described in the Materials and Methods. Forhuman MLR cultures the same 3 different responder preparations were usedfor each Mab, and stimulated with a pool of mitomycin C treated spleenstimulator cells (from a random mixture of 6 spleen donors). For mouse(C3H anti-C57BL/6) and rat (LEW anti-BN) MLR cultures all assays wereset up in triplicate for each Mab. Mouse responder spleen cells werefrom mice treated 4 days earlier by portal vein infusion of C57BL/6 bonemarrow cells, except for data shown as (None**) where responder cellswere from non-injected C3H mice. Mab was added as a 30% superntatantconcentration. Supernatants were harvested for cytokine assays at 60hrs. ^(b)Data show arithmetic means (+SD) for each Mab. For mouse assaysall supernatants were assayed for a number of cytokines (ELISA), and forIL-2/IL-6 using bioassays (proliferation of CTLL-2, B9 respectively).Supernatants from rat/human cultures were assayed in bioassays only.Note that cells incubated with isotype control Igs (non-reactive byELISA or FACS) gave cytokine data indistinguishable from culturesincubated in the absence of Mab. p < 0.05, compared with cultureswithout Mabs.

TABLE 5 OX-2:FC Immunoadhesin Inhibits Mixed Leukocyte Reaction in vitroCytokines in Added Percent lysis ⁵¹Cr targets^(b) culture (pg/ml)^(c)supernatant^(a) (50:1, effector:target) IL-2 IL-4 NONE (control) 31 ±4.0 1005 ± 185 60 ± 20 Control CHO 33 ± 4.3  810 ± 190 45 ± 20 (vectortransduced) CHO transduced 4.2 ± 2.1  175 ± 45 245 ± 55  with OX-2:FcFootnotes: ^(a)Supernatant was harvested at 7 days from CHO cellstransduced with control pbK vector, or vector carrying a cDNA insertencoding OX-2 linked to murine Fc. A 1:1 mixture of supernatant was usedin cultures containing 5 × 10⁶ LEW spleen and 2.5 × 10⁶ irradiated LBNF1spleen cells; this corresponded to 50 ng/ml OX-2:Fc ^(b)and ^(c)Percentlysis with cells at 5 days, using 1 × 10⁴ ⁵¹Cr BN spleen ConA targets;cytokines in culture supernatants at 60 hrs.

TABLE 6 Inhibition of skin graft rejection by OX-2:Fc Rejection of skingrafts Treatment of mice (mean + SD) in days NIL 12 + 3.8 OX-2:Fc 19 +4.2 Footnotes: 6 mice/group were treated as shown. NIL indicatesinfusion of normal mouse IgG only. Arithmetic mean (+SD) graft survivalfor group.

TABLE 7 OX-2:Fc infused into mice receiving skin allografts reversespolarization in cytokine production Cytokines in culture supernatant at48 hrs (pg/ml) Treatment of mice IL-2 IL-4 NIL 1250 + 160  80 + 20OX-2:Fc 350 + 85 245 + 50 Footnotes: 3 mice/group received iv infusionsof saline or OX-2:Fc (50 mg/mouse) every 2 days ×4 from the time ofgrafting with C57BL/6 skin. Mice were sacrificed at 10 days and spleencells stimulated in vitro with irradiated C57BL/6 spleen stimulatorcells. Arithmetic mean (+SD) for IL-2/IL-4 in supernatant at 48 hrs.Data are pooled from triplicate cultures for each mouse spleen.

TABLE 8 Preimmunization of mice with EL4-CD86 causes increased CD200expression which leads to generalized suppression to newly encounteredalloantigen Tumor used for Cytokines in Supernatant^(c) Immunization^(a)% Lysis^(b) IL-2 IL-4 IFNγ IL-10 NONE (control) 43 ± 5.5 980 ± 125 50 ±10 455 ± 65 35 ± 10 EL4 41 ± 6.2 890 ± 135 60 ± 15 515 ± 70 30 ± 10EL4-CD80 46 ± 6.3 955 ± 140 45 ± 15 525 ± 55 30 ± 10 EL4-CD86  16 ± 4.2*420 ± 75* 125 ± 20*  240 ± 40* 120 ± 20* NONE (control)+ 46 ± 5.8 950 ±105 55 ± 15 490 ± 60 40 ± 10 EL4+ 44 ± 4.9 940 ± 115 50 ± 20 530 ± 60 35± 10 EL4-CD80+ 44 ± 6.0 905 ± 120 60 ± 15 555 ± 75 35 ± 10 EL4-CD86+  39± 4.29 870 ± 125 75 ± 20 540 ± 65 40 ± 15 Footnotes: ^(a)Spleen cellswere pooled from 3 C57BL/6 mice/group, pretreated as described in thetext, by immunization with 5 × 10⁶ mitomycin-C treated EL4 tumor cells,or CD80/CD86-transfected tumor cells, in Complete Freund's Adjuvant 4days earlier. 5 × 10⁶ spleen cells were incubated in triplicate withequal numbers of mitomycin-C treated BALB/c spleen stimulator cells. +indicates anti-CD200 (anti-OX2) added to cultures (5 μg/ml) ^(b)%specific lysis in 4-hr ⁵¹Cr release assays with 72-hr cultured BALB/cspleen Con A blast cells (effector:target ratio shown is 100:1).^(c)Cytokines in culture supernatants assayed in triplicate by ELISA at40 hrs (see Materials and Methods). Data represent pg/ml except forIL-10 (ng/ml). *p < 0.05 compared with all groups.

TABLE 9 Mice Experiment Mouse type: Swiss-Webster-Outbred Test Article:Clone 3B6rat - anti-mouse OX-2 Dose 100 pg IP Q 3 days Placebo: MouseIgg Mouse Date Delivery Number Plugged Date Litter size Controls 1 28Feb. 2001 Mar-21 8 2 Feb-29 Mar-21 6 3 Feb-29 Mar-21 8 4 Mar-01 Mar-22 65 Mar-01 Mar-22 6 6 Mar-03 Mar-24 8 7 Mar-03 Mar-24 4 8 Mar-05 Mar-26 59 Mar-05 Mar-26 8 10 Mar-06 Mar-27 8 11 Mar-06 Mar-27 6 12 Mar-06 Mar-288 13 Mar-08 Mar-29 8 14 Mar-08 Mar-29 6 15 Mar-08 Mar-30 4 16 Mar-08Mar-30 8 17 Mar-11 Mar-31 6 Treated 1 Feb-28 Mar-20 7 2 Feb-29 Mar-21 83 Feb-29 Mar-21 8 4 Feb-29 Mar-21 4 5 Mar-01 Mar-22 5 6 Mar-03 Mar-22 87 Mar-03 Mar-23 8 8 Mar-05 Mar-25 6 9 Mar-05 Mar-25 6 10 Mar-06 Mar-25 511 Mar-06 Mar-26 4 12 Mar-06 Mar-27 4 13 Mar-06 Mar-27 8 14 Mar-08 Nil 015 Mar-08 Mar-29 6 16 Mar-08 Mar-29 7 17 Mar-08 Mar-29 8 18 Mar-11Mar-31 7 Controls % Pregnant 100 Treated: % Pregnant 94

TABLE 10 Mice Experiment Mouse type: Swiss-Webster-Outbred Test Article:Clone 3B6rat - anti-mouse OX-2 Dose 250 micrograms IP every 36 hoursPlacebo: Mouse Igg Litter Mouse Delivery Litter Size Litter Size SizeNumber Date Plugged Date Males Females Total Controls 1 Apr-01 Apr-21 42 6 2 Apr-01 Apr-22 4 3 7 3 Apr-03 Apr-25 3 3 6 4 Apr-05 Apr-26 2 4 6 5Apr-05 Apr-27 5 3 8 6 Apr-08 Apr-29 4 4 8 7 Apr-09 Apr-30 3 3 6 8 Apr-10Apr-30 2 4 6 Treated 1 Apr-01 Nil 0 0 0 2 Apr-02 Nil 0 0 0 3 Apr-04Apr-25 4 2 6 4 Apr-05 Nil 0 0 0 5 Apr-07 Apr-29 1 3 4 6 Apr-09 Apr-30 42 6 7 Apr-10 Nil 0 0 0 8 Apr-13 Nil 0 0 0 Controls % Pregnant 100Treated % Pregnant 38

TABLE 11 Rat Experiment Rat type: Sprague-Dawley-Outbred Test Article:Clone 6C2 Mouse - anti-rat OX-2 Dose 300 micrograms IP Q 3 days Placebo:Rat Igg Controls RAt Date Delivery Number Plugged Date Litter size 1Feb-29 Mar-21 11 2 Feb-29 Mar-22 13 3 Mar-01 Mar-24 10 4 Mar-01 Mar-2411 5 Mar-03 Mar-25 12 6 Mar-03 Mar-25 9 7 Mar-05 Nil 0 8 Mar-05 Mar-27 99 Mar-06 Mar-28 8 10 Mar-07 Mar-28 9 11  Mar-07 Mar-29 10 12  Mar-09Mar-31 8 Treated Rat Date Delivery Number Plugged Date Litter size 1Feb-29 Mar-21 8 2 Mar-01 Mar-23 9 3 Mar-01 Nil 0 4 Mar-01 Mar-24 9 5Mar-03 Nil 0 6 Mar-05 Nil 0 7 Mar-06 Mar-28 8 8 Mar-07 Mar-29 6 Controls% Pregnant 92 Treated: % Pregnant 63

TABLE 12 Rat Experiment Rat type: Sprague-Dawley-Outbred Test Article:Clone 6C2 Mouse - anti-rat OX-2 Dose 500 micrograms IP Q 3 days Placebo:Rat Igg Controls Litter Litter RAt Date Delivery Litter Size Size SizeNumber Plugged Date Males Females Total 1 Apr-04 Apr-25 6 4 10 2 Apr-04Apr-27 6 5 11 3 Apr-08 Apr-29 4 6 10 4 Apr-09 Apr-30 3 7 10 5 Apr-13May-02 4 5 9 6 Apr-14 May-03 6 5 11 Treated Litter Litter Rat DateDelivery Litter Size Size Size Number Plugged Date Males Females Total 1Apr-05 Nil 0 0 0 2 Apr-06 Nil 0 0 0 3 Apr-09 Apr-30 3 4 7 4 Apr-12 Nil 00 0 5 Apr-14 Nil 0 0 0 6 Apr-16 May-06 4 4 8 Controls % Pregnant 100Treated: % Pregnant 33

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1. A method of inhibiting type-1 cytokine production comprisingadministering an effective amount of a CD200 protein or a CD200 proteinfragment that comprises an extracellular domain of a CD200 protein to ananimal in need thereof.
 2. A method according to claim 1 wherein theCD200 protein is a human CD200 protein or a fragment thereof.
 3. Amethod according to claim 1 wherein the CD200 protein is a solublefusion protein.
 4. A method according to claim 3 wherein the solublefusion protein comprises a CD200 protein or fragment thereof linked toan immunoglobulin Fc region.
 5. A method according to claim 1 to treatautoimmune disease.
 6. A method according to claim 5 wherein theautoimmune disease is selected from the group consisting of arthritis,type 1 insulin-dependent diabetes mellitus, adult respiratory distresssyndrome, inflammatory bowel disease, dermatitis, meningitis, thromboticthrombocytopenic purpura, Sjogren's syndrome, encephalitis, uveitis,leukocyte adhesion deficiency, rheumatoid arthritis, rheumatic fever,Reiter's syndrome, psoriatic arthritis, progressive systemic sclerosis,primary biliary cirrhosis, pemphigus, pemphigoid, necrotizingvasculitis, myasthenia gravis, multiple sclerosis, lupus erythematosus,polymyositis, sarcoidosis, granulomatosis, vasculitis, perniciousanemia, CNS inflammatory disorder, antigen-antibody complex mediateddiseases, autoimmune haemolytic anemia, Hashimoto's thyroiditis, Gravesdisease, habitual spontaneous abortions, Reynard's syndrome,glomerulonephritis, dermatomyositis, chronic active hepatitis, celiacdisease, tissue specific autoimmunity, degenerative autoimmunity delayedhypersensitivities, autoimmune complications of AIDS, atrophicgastritis, ankylosing spondylitis and Addison's disease.
 7. A methodaccording to claim 5 wherein the autoimmune disease is arthritis.
 8. Amethod according to claim 1 to inhibit or reduce fetal loss.
 9. A methodaccording to claim 1 to suppress an immune response to a transplantedtissue, cell or organ.
 10. A method according to claim 1 to treat anallergy.
 11. A method according to claim 1 wherein said CD200 protein orfragment is capable of inhibiting a type-1 cytokine selected frominhibiting interleukin-2 production and inhibiting interferon-γproduction.
 12. A method according to claim 5 wherein the soluble fusionprotein comprises a CD200 protein or fragment thereof linked to animmunoglobulin Fc region.
 13. A method according to claim 8 wherein thesoluble fusion protein comprises a CD200 protein or fragment thereoflinked to an immunoglobulin Fc region.
 14. A method according to claim 9wherein the soluble fusion protein comprises a CD200 protein or fragmentthereof linked to an immunoglobulin Fc region.
 15. A method according toclaim 10 wherein the soluble fusion protein comprises a CD200 protein orfragment thereof linked to an immunoglobulin Fc region.